Contraceptive vaccine based on alloimmunization with zona pellucida polypeptides

ABSTRACT

The present invention relates to contraceptive vaccines based on cloned zona pellucida genes and the strategy of alloimmunization with zona pellucida polypeptides. In particular, the present invention relates to a contraceptive vaccine for use in a mammalian female comprising a polypeptide which displays at least one epitope for binding of an antibody that inhibits fertilization of an oocyte by a sperm. This epitope is from a zona pellucida procein of the species in which the said vaccine is used. This invention relates, more particularly, to such vaccines wherein the zona pellucida protein is either the ZP3 or the ZP2 or the ZP1 protein or the mouse or homologues of these proteins in some other mammalian species. Further, this invention comprehends vaccines comprising a synthetic peptide that displays an epitope for such an antibody that inhibits fertilization. In addition, this invention relates to cloned DNA segments variously encoding the mouse ZP3 or ZP2 proteins or the human ZP3 or ZP2 protein.

This application is a divisional of Ser. No. 08/453,952, filed May 30,1995, now U.S. Pat. No. 5,672,488; which is a divisional of Ser. No.08/038,948, filed Mar. 26, 1993, now U.S. Pat. No. 5,641,487; which is acontinuation-in-part of Ser. No. 07/930,642, filed Aug. 20, 1992, nowabandoned; which is a continuation of Ser. No. 07/364,379, filed Jun.12, 1989, now abandoned.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to contraceptive vaccines based dn clonedzona pellucida genes and the strategy of alloimmunization with zonapellucida polypeptides. In particular, the present invention relates toa contraceptive vaccine for use in a mammalian female comprising apolypeptide which displays at least one epitope for binding of anantibody that inhibits fertilization of an oocyte by a sperm. Thisepitope is from a zona pellucida protein of the species in which thesaid vaccine is used.

This invention relates, more particularly, to such vaccines wherein thezona pellucida protein is either the mouse ZP2 protein, the mouse ZP3protein, the human ZP2, the human ZP3 protein, or homologues of theseproteins found in other mammalian species. Further, this inventionincludes vaccines comprising a synthetic peptide that displays anepitope for such an antibody that inhibits fertilization. In addition,this invention relates to cloned DNA segments variously encoding themouse ZP3 or ZP2 proteins, or the human ZP2 or ZP3 proteins.

2. Background Information

There is currently much interest in the development of a safe andeffective contraceptive vaccine for control of diverse mammalianpopulations. Contraceptive vaccines would be useful under certaincircumstances where relatively long-term but not permanent contraceptionis desired without the need for frequent intervention, for example, inpets including cats and dogs, in agriculturally important livestock suchas cattle and pigs, and in human beings. A contraceptive vaccinepreferably should have an effect which is long-lasting and highlyspecific. Further, to minimize possibilities for birth defects in theevent of failed contraception, the antigen which is selected as theimmunogen should produce contraceptive antibodies that inhibitfertilization of the egg by a sperm rather than by an abortifacientmechanism involving disruption of early development. In addition, thevaccine preferably should induce an immunological response that issufficient to be effective for contraception without eliciting acytotoxic response that might result in abnormal reproductive function.

The mammalian zona pellucida, which surrounds growing oocytes andovulated eggs, has been recognized as a potential immunogen for acontraceptive vaccine (C. J. Henderson, et al., J. Reprod. Fert. 83:325-343 (1988); B. S. Dunbar, 1983, Mechanisms and Control of AnimalFertilization, J. F. Hartmann, ed., pp. 140-175, Academic Press, NewYork; A. T. Sacco, Am. J. Reprod. Immunol. Microbiol. 15: 122 (1987);Millar et al., Targeting of zona pellucida for immunocontraception, inImmunology of Reproduction, Naz, R. K. (ed.), pp. 293-313 (1993)). Atbirth the mouse ovary contains 10,000-15,000 oocytes in the prophase ofthe first meiotic division. As cohorts (10-15) of these oocytes enterinto a two week growth phase, they synthesize and secrete zona proteinsto form the extra-cellular zona pellucida which ultimately reaches athickness of 7 μm in the fully grown oocyte. The zona is unique to theovary, being highly antigenic and accessible to circulating antibodyduring the two week intra-ovarian oocyte growth phase prior to meioticmaturation and ovulation.

Passive immunization of mice or hamsters with anti-zona sera has beenshown to produce reversible contraception without obvious side effects.For example, U.S. Pat. No. 3,992,520 to Gwatkin discloses, inter alia,an anti-serum composition for short-term control of fertility comprisingantibody obtained by immunizing an animal with water solubilized zonapellucida of a distinct donor species. This method requires isolation oflarge amounts of a relatively scarce natural antigen which would not befeasible for certain mammals such as humans. Further, long-termadministration of antibodies from a foreign (i.e., "heterologous")species leads to induction of reactive antibodies that will inhibit thecontraceptive action of the contraceptive antibodies. Further,administration of serum or products isolated from serum carries inherentrisks of transmission of blood-born diseases.

Structural information about the zona pellucida has been available forsome years. The mouse zona, for instance, is composed of three sulfatedglycoproteins, designated ZP1, ZP2 and ZP3, (J. D. Bleil et al., Dev.Biol. 76:185 (1980); S. Shimizu et al., J. Biol. Chem. 258:5858 (1983))which play important roles in fertilization and early development andhave average M_(r) s of 200,000, 140,000, and 85,000, respectively. ZP2and ZP3 appear to be complexed into long filaments which arecross-linked by ZP1 in the zona matrix providing structural integrity tothe zona pellucida. Sperm initially bind to ZP3 via O-linkedoligosaccharide chains and continued binding involves ZP2 as a secondarysperm receptor. Subsequently, ZP3 induces lysis of the sperm's acrosomewhich releases enzymes (such as glycosidases and proteases) which arethought to be important for the penetration of the zona pellucida bysperm. Following fertilization, both ZP2 and ZP3 are biochemicallymodified to prevent additional sperm binding and thereby to facilitatethe post-fertilization block to polyspermy.

The zona pellucida in other mammals besides the mouse is known tocomprise several distinct glycoproteins components with apparent sizesand, hence naming terminologies, that do not necessarily corresponddirectly to the mouse ZP1 (185-200 kDa), ZP2 (120-140 kDa) and ZP3 (83kDa) proteins. The human zona pellucida is composed of three proteinsdesignated ZP1 (90-110 kDa), ZP2 (64-76 kDa) and ZP3 (57-73kDa)(Shabanowitz et al., J. Reprod. Fertil. 82:151-61 (1988);Shabanowitz, 43:260-70 (1990)) and other species in which zona proteinshave been characterized include hamster (Moller et al., 137:276-86(1990), pig (Dunbar et al., Biol. Reprod. (1981); Hedrick et al., Dev.Biol. 121:478-88 (1987); Yurewicz et al., J. Biol. Chem. 262:564-71(1987), rabbit (Dunbar et al., Biol. Reprod. 24:1111-24 (1981) and horse(Millar et al., J. Reprod. Fert. 96:815-25 (1992)). The correspondenceof specific zona proteins among different species is becoming clearer asadditional information on the primary amino acid sequence is deducedfrom cloned zona pellucida genes (Ringuette et al., Proc. Natl. Acad.Sci. U.S.A. 83:4341-45 (1986); Ringuette et al., Dev. Biol. 127:287-95(1988); Chamberlin et al., Proc. Natl. Acad. Sci. U.S.A. 87:6014-18(1990); Chamberlin et al., Dev. Biol. 131:207-14 (1989); Liang et al.,Mol. Cell. Biol. 10:1507-15 (1990); Liang et al., Dev. Biol. 156:399-408(1993); Kinloch et al., Dev. Biol. 142:414-21 (1988); Schwoebel et al.,J. Biol. Chem. 266:7214-19 (1991); Kinloch et al., Dev. Biol. 142:414-21(1990)) and direct sequencing of peptides derived from zona pellucidaproteins (Ringuette et al., supra (1986); Yurewicz et al., Mol. Reprod.Dev. 33:182-88 (1992)).

In light of the identification of the distinct murine zona pellucidapolypeptides, ZP1, ZP2 and ZP3, further experiments on passiveimmunization with contraceptive antibodies have been conducted.Specifically, rat anti-mouse ZP2 and anti-mouse ZP3 monoclonalantibodies were injected into female mice and were found to bindspecifically to the zonae surrounding growing, intra-ovarian oocytes.After ovulation, the binding of the antibody to the zona persisted; andthe presence of these antibodies precluded fertilization by preventingsperm from penetration of the zona pellucida. This contraceptive effectwas long-term, lasting approximately 15 mouse estrus cycles, but waseventually reversible. There was no evidence of any adverse effect onthe development of fertilized embryos to term and no evidence ofabnormal ovarian histology or function. However, the antibody bindingsites (i.e., "epitopes") recognized on mouse ZP2 and ZP3 by fivedifferent rat anti-mouse monoclonal antibodies that were tested are notpresent on other mammalian zonae pellucidae (East et al., J. Cell Biol.98:795-800 (1984); East et al., Dev. Biol. 104:49-56 (1984); and East etal., Dev. Biol. 109: 268-73 (1985)). This species specificity limits theusefulness of these particular antibodies as contraceptive agentsessentially to murine species. In addition, even if analogous murineanti-ZP2 or anti-ZP3 antibodies that inhibit fertilization could beidentified for ZP2 or ZP3 of non-murine species, there are inherentside-effects from the repeated administration of heterologousantibodies, as noted above.

There have been several studies on active immunization usingpreparations of isolated zona pellucidae to immunize rodents, rabbits,and primates (C. J. Henderson, et al., J. Reprod. Fert. 83:325 (1988);R. B. L. Gwatkin, et al., 1977, Fert. Steril. 28:871 (1977); Drell etal., Biol. Reprod. 30:435-44 (1984); Sacco et al., Biol. Reprod.36:481-90 (1987); Jones et al., J. Reprod. Fertil. 95:513-25 (1992)).

Further, the U.S. Patent to Gwatkin cited above (U.S. Pat. No.3,992,520) also discloses a vaccine for the immunological control offertility in female mammals that consists of an aqueous solution ofwater solubilized zona pellucida prepared by heating mammalian zonepellucida at 65-100° C. in an aqueous medium. One example thereindescribes a bovine antigen preparation intended for use in humans.

U.S. Pat. No. 4,996,297 of Dunbar is limited to three rabbit cDNAsequences S1, P2, and P3 thought to encode rabbit zona proteins, to theuse of these cDNAs to produce polypeptides that contain epitopes onthree rabbit zona proteins (50 kDa, 75 kDa, and 80 kDa), and to the useof the recombinant polypeptides to vaccinate other mammals in order toelicit antibodies that bind to that mammal's zona pellucida forcontraception (i.e., heteroimmunization).

Japanese Patent 63,150,299 discloses a pig zona pellucida antigen foruse as a contraceptive vaccine for pigs or humans that is characterizedas a glycoprotein of 20 to 30 kDa in molecular weight which can beextracted from soluble pig zona pellucida with 8.5M urea and 2%2-mercaptoethanol.

Despite positive results under experimental conditions, methods ofpreparing a vaccine from natural zona pellucida materials are clearlydifficult if not outright impractical for commercial use, particularlyin the human case, due to limited sources of antigen and to difficultiesin quality control of such poorly defined vaccines. Further, wide-spreadovarian histopathology and dysfunction were reported in rabbits, dogsand primates after active immunization with zonae pellucidae orextracted antigens (see, for example, R. B. L. Gwatkin, et al., GameteRes. 1:19 (1980); A. T. Sacco, Am. J. Reprod. Immunol. Microbiol. 15:122(1977)). Several studies have suggested that both the dose and thepurity of the immunogen contributed to these abnormalities, twoproperties that are particularly difficult to control in such relativelycrude antigen preparations.

The effect of the genetic origin of the zona pellucida antigen on itsability to immunize a given species against conception has been examinedin several studies. For instance, the efficacies of contraceptiveimmunizations with pig and rabbit zonae pellucidae on fertility inrabbits was compared. This comparison of results with "alloimmunization"(literally "self-immunization", using antigen from the same species,i.e., an "alloantigen") with those of "heteroimmunization" (usingantigen from another species, i.e., an "heterologous" antigen) suggested(D. M. Wood et al., Biol. Reprod. 25:439-450 (1981)) thatheteroimmunization of rabbits with porcine zonae is more effective inreducing fertility than alloimmunization with rabbit zonae. More recentwork using immunoaffinity purified antibodies to zona pellucida tocompare immune responses in alloimmunization of male and female rabbitshas continued to support the greater effectiveness for contraception ofheteroimmunization with zona pellucida antigens. (S. M. Skinner, et al.,J. Reproductive Immunology 12:81-92 (1987)).

Another general approach toward providing a vaccine related to anyantigen involves the use of a particular type of antibody, called an"anti-idiotypic" antibody, as an immunogen to actively immunize ananimal. Anti-idiotypic antibodies are antibodies directed to the antigenbinding site of another antibody; accordingly, the antigen binding siteof the anti-idiotypic antibody mimics or represents an image of the siteon the antigen that is bound by the other antibody. U.S. Pat. No.4,795,634 to Grimes et al. (equivalent of WO 87/05,516) discloses avaccine that comprises anti-idiotypic antibodies to anti-zona pellucidaantibodies to express images of zona pellucida antigens. This vaccinesuffers from drawbacks including the fact that anti-idiotypic antibodiesare generally difficult and expensive to prepare in amounts and puritysatisfactory for vaccine usage, particularly in human applications.Further, heteroimmunization with antigens comprising antibodies fromanother species may induce predominantly antibodies to sites on theantibody other than the desired target, the antigen binding site. Inother words, the desired antigen binding site may not constitute an"immunodominant" antigenic site (or "determinant") for the vaccineantibody protein in a species different from that which produced thevaccine protein (see below for a discussion on the basis ofimmunodominance). (See also U.S. Pat. No. 4,996,297 of Dunbar et al.)

Another technique for producing vaccines that is known generally in theart is the use of specific isolated polypeptides as antigens, or ofpeptides representing portions of such polypeptides, in place of crudeantigen preparations comprising aqueous extracts of target tissues.Accordingly, European Patent EP-0117934 to Stevens discloses a modifiedantigen for use in fertility control comprising an unspecified antigenfrom the zona pellucida, or a peptide having a sequence corresponding toat least part of the sequence of such a zona pellucida antigen, whichantigen or peptide has been chemically modified outside the body of theanimal. The modified antigen has a greater capacity to induce antibodiesthan the unmodified antigen from which it is derived. According to thespecification and claims, such modification includes coupling theantigen or peptide through a maleimido linkage to a suitable "carrier"protein that is biologically foreign to the animal to be vaccinated andof size sufficient to elicit antibody response. Neither this Europeanapplication nor any related applications, as yet published, teachesspecific zona pellucida polypeptides or peptides that are suitable foruse as contraceptive vaccines.

In light of the complexities, difficulties and uncertainties of all thecontraceptive vaccines described above, there is yet a need for asimpler, safer, cheaper, more defined and effective contraceptivevaccine. The present invention is based on the premise that vaccinationwith a "self" zona protein (alloimmunization) is most likely to elicitantibodies that will cross-react with the native zona pellucida andprevent fertilization. Furthermore, by using relatively short peptidesas immunogens, the adverse effects on ovarian structure and functions,at least some of which can result from a T cell mediated autoimmuneresponse, can be avoided. However, the success of this approach dependson knowledge of the primary amino acid sequence of the zona pellucidaproteins. Because of the paucity of biological material, this sequenceinformation can only be obtained by cloning cDNAs encoding the zonaproteins and deducing the amino acid sequence from the nucleic acidsequence. Toward this end, the present inventor and associates haverecently constructed a mouse ovarian cDNA expression library andisolated two overlapping ZP3 cDNA clones (M. J. Ringuette et al., Proc.Natl. Acad. Sci. USA 83:4341 (1986)), one of which expresses a fusionprotein recognized by an anti-ZP3 monoclonal antibody (East et al., Dev.Biol. 109: 268 (1985)).

The identity of these clones was confirmed by a comparison of the aminoacid sequence encoded by a 60 nucleotide stretch of their nucleic acidsequence with the terminal amino acid sequence (20 amino acids) of alarge internal fragment isolated from the ZP3 protein (Ringuette et al.,supra 1986)). This fragment was isolated from purified ZP3, followingdigestion with a protease, by affinity chromatography using an anti-ZP3monoclonal antibody. Therefore, it was clear that this fragment wascapable of expressing an epitope for a contraceptive antibody; however,the location of that epitope within scores of amino acid residues wasnot known, and as disclosed herein, is distinct from the 20 amino acidsequence obtained. More importantly, the ability of this proteolyticcleavage fragment to serve as an immunogen in a vaccine was not known,nor was there any practical means for preparing sufficient material fromnatural sources to test that cleavage fragment further.

A first attempt to utilize the cloned mouse ZP3 cDNA described above toproduce a vaccine was unsuccessful (S. M. Chamow and J. Dean, 1987,abstract of presentation to the American Society of BiologicalChemists). This effort involved testing of the recombinantZP3-β-galactosidase fusion protein, which contained most of the ZP3amino acids as well as a larger portion of β-galactosidase and wasgenerated according to well known methods in genetic engineering thathave successfully produced other antigens with native immunoreactivity.Immunization with this particular fusion protein, however, failed toinduce detectable antibodies that would react with native ZP3;reactivity was detected only after reduction of disulfide bonds anddenaturation.

The basis of this failure to induce anti-ZP3 contraceptive antibodies,despite that fact that the cDNA clearly encoded a proteolytic cleavagefragment that reacted with such an antibody, is not entirely clear. Itmay be that, under the conditions of immunization, the portion of thefusion protein that encoded the contraceptive antibody epitope did notassume the proper conformation to react with such antibodies. In otherwords, although the fusion protein surely encoded the amino acids thatformed the epitope in the native ZP3 protein, it may be that those aminoacids did not exhibit (i.e., did not "display") that epitope in thisinstance. It is also possible that epitopes for other antibodies, whichwere located on the β-galactosidase moiety of the fusion, may have beenimmunodominant over the contraceptive antibody epitopes and thusprevented a detectable contraceptive antibody response (see discussionof immunodominance below). Finally, a combination of these effects andothers may have united to prevent the desired contraceptive antibodyresponse to the fusion product of the recombinant DNA which expressedmost of the ZP3 polypeptide. These results clearly illustrate theunpredictability of the immunogenicity of a polypeptide under any givenset of conditions, no matter how efficacious they may be for otherantigens, and the need for experimental determination of the necessaryphysical form of the amino acids that encode an epitope (e.g.,polypeptide size and nature of attached amino acid sequences) to displaythat epitope and, further, to induce antibodies to it.

Accordingly, it is an object of the present invention to find anefficacious way to use contraceptive antibodies and cloned genesencoding zona pellucida proteins to develop contraceptive vaccines foruse in a mammalian female. More particularly, it is an object of thisinvention to provide such vaccines comprising polypeptides that includedefined amino acid sequences that are selected for their ability todisplay epitopes for contraceptive antibodies.

Additional immunological analyses of the individual ZP polypeptidecomponents have been carried out. For example, specific monoclonal andpolyclonal antibodies have been employed to define distinct antigens ofthe porcine zonae pellucidae, leading to the suggestion that there areboth unique and shared antigenic determinants present in the individualcomponents of the zona pellucida, but that the immunodominantdeterminants appear to be unique to each glycoprotein (T. M. Timmons, etal., Biology of Reproduction 36:1275-1287 (1987)).

Finally, there has been a report of an effort to molecularly clone cDNASencoding specific antigenic sites from rabbit ZP proteins usingantibodies that recognize determinants found on ZP antigens of severalspecies (P. Cheung et al., 1987, abstract of a presentation at thetwenty-seventh annual meeting of the American Society for Cell Biology,St. Louis, Mo., November 16-20, J. Cell Biol. 105, no. 4 part 2, 334A).This abstract reported in part that:

"These studies demonstrated that cross-species affinity purification ofantibodies is an effective method for isolating cDNA clones expressingantigens which are shared among different mammalian species."

However, no specific nucleotide or amino acid sequences were disclosedin this abstract, nor was the contraceptive potential of the antibodiesdiscussed; indeed, there was no mention of any contraceptive vaccine.

In a speculative exposition on the use of recombinant DNA and syntheticpeptide technologies for development of a human contraceptive vaccinefrom porcine zona pellucida antigens (C. J. Henderson, et al., J.Reprod. Fert. 83:325 (1988)), the identification of amino acid sequencesdisplaying epitopes for contraceptive vaccines on a particular porcinepolypeptide is anticipated, although absolutely no sequences of thepolypeptide are disclosed. Nevertheless, this reference goes on tohypothesize that known vaccine technologies, including syntheticpeptides and vaccinia virus expression vectors, will provide successfulhuman vaccines based on this particular porcine polypeptide that isknown to be immunologically related to human zona pellucida antigens.Furthermore, while asserting that monoclonal antibodies to thispolypeptide that exert a contraceptive effect "will be extremelyimportant in defining the epitopes with contraceptive potential . . .",this report also notes that, despite obtaining monoclonal antibodiesreactive with this polypeptide, the authors "have failed to generate amonoclonal antibody with contraceptive effect; this is in accord withother published reports . . . ."

Although a complete exposition of the current theoretical basis ofimmunogenicity and antigenicity of polypeptides is beyond the scope ofthe present disclosure, a brief discussion of selected principles andterms of this active art will facilitate further understanding of theinstant invention. In this application, absent an express statement tothe contrary, each use of the term "polypeptide" encompasses any polymercomprising two or more amino acids coupled by peptide linkages (i.e.,dipeptides, oligopeptides, peptides, polypeptides) as well as proteinsconsisting of multiple polypeptide subunits.!

Accordingly, it should be noted, first, that the necessary andsufficient properties of a polypeptide for inducing antibodies cannot bepredicted for any given set of conditions (e.g., for a particularspecies, or for presentation in a certain form). Nevertheless, much morehas been learned about this subject in the past decade than is reflectedin any of the art cited so far herein, and it is a further object of thepresent invention to exploit aspects of this knowledge for design ofadvantageous contraceptive vaccines.

In particular, comprehension of the present invention will be aided bythe now widely held view that the nature and level of the immuneresponse to a polypeptide depends on its interactions with at least twodistinct classes of immune system cells, namely B-cells and T-cells. Insimple terms, the role of B-cells in immunity may be thought of asrecognition of the specific sites on macromolecules to which antibodiesare produced and subsequent production of those antibodies. These B-cellrecognition sites, which provide the main basis for immune recognitionof non-self molecules and are also called B-cell epitopes, are of a sizecorresponding to about that of the antigen binding site on an antibody,typically of a diameter equivalent to the length of a peptide containingabout four to six amino acids.

It may be noted here that there exists a formal distinction between theepitope for a B-cell and that of its related antibody. In other words,due to complex biological mechanisms that intervene between therecognition by a B-cell of a given site on an antigen and the consequentproduction of antibodies to that site, it is possible that the ultimateantibody recognition site may not be precisely identical to theinitially recognized B-cell epitope. However, for the present purposes,a B-cell epitope may be considered to be essentially the same structureas the binding site for the corresponding antibody.!

The functions of T-cells, on the other hand, relate in large measure tohelping to activate antibody production by B-cells upon initial exposureto an antigen, as well as to enhancing their antibody response uponsubsequent reexposures (i.e., to "immune memory" or the "amnestic"response). To play their roles in immunity, T-cells must also recognizespecific sites on an antigen to which antibodies are produced, and suchT-cell epitopes are about the same size as B-cell epitopes.

B-cell and T-cell epitopes on any given polypeptide, however, need notcomprise the same amino acid residues. In fact, it will be appreciatedby those of ordinary knowledge in the current art of peptide immunologyat the molecular level, that even in a peptide consisting of only half adozen amino acids, there may coexist several different B-cell epitopes(comprising, for instance, from two to four atoms that contactcomplementary structures on the antibody) and one or more distinctT-cell epitopes which may or may not include atoms of amino acids alsoincluded in a B-cell epitope.

It is also well known that the vast majority of small peptides(containing six to twenty amino acids, for instance) that have beentested for induction of antibodies are considerably less potentimmunogens than the larger proteins from which they have been derived,despite ample ability of the peptides to bind to antibodies directedagainst those larger proteins. Certain chemical modifications of apeptide, particularly coupling of the peptide to a larger proteinaceous"carrier", generally enhance the immune response to a small peptide.

Although the role of such a carrier still may not be fully understood inall respects, it has been clearly established, in particular, that thereis no specific minimum size requirement for peptides in general toinduce a substantial immune response. Rather, it is now widely believedthat a major function of the carrier is to provide T-cell epitopes inclose association with the B-cell epitopes on the short peptide which isstatistically unlikely to contain both T-cell and B-cell sitesrecognized by the immune system of any given individual.

It may also be noted here that it has been shown that a T-cell epitopetaken from one protein, in the form of a short peptide, may be combinedwith a short peptide comprising a B-cell epitope of another protein, toform a single peptide that induces a more complete and higher levelimmune response than either peptide alone.

More broadly, it is now widely accepted that the capability of anyindividual to mount any immune response to a given epitope, as definedby a precise configuration of a small number of atoms, dependsultimately on the genetic make-up of the immune system genes whichseparately control the specificities of antigen recognition by B-cellsand T-cells. Further, it is understood that the ability of a givenB-cell epitope to induce cognate antibodies (i.e., antibodies whichrecognize that epitope) also depends upon the context within which thatepitope is presented to the immune system, in terms of both associatedT-cell epitopes and other B-cell epitopes. The latter sites may be"immunodominant" relative to the selected B-cell epitope of interest,that is, they may contend more effectively for the attention of theimmune system than the selected B-cell epitope and thereby distractlimited system resources from mounting the desired response to thatselected epitope. In other words, B-cell epitopes that do not inducedetectable antibodies in the presence of other, so-called immunodominantepitopes, which frequently occur in large polypeptides, often do inducesignificant levels of cognate antibodies when presented in a differentcontext that lacks such immunodominant sites, on a short peptide, forexample.

In conclusion, it is a further object of the present invention toexploit various consequences of the above noted characteristics of anddistinctions between B-cell and T-cell epitopes, as well as methods forpredicting and actually detecting amino acid sequences that serve asT-cell or B-cell epitopes. These will be discussed further below, asneeded, in relation to the description of the present invention.

SUMMARY OF THE INVENTION

The recent molecular cloning, by the present inventor, of DNA segmentscorresponding to the mouse ZP3 and ZP2 genes, the human ZP3 and ZP2genes, and the subsequent characterization of the nucleotide sequencesof their messenger RNAs (mRNAs) and the amino acid sequences encodedthereby, have provided sufficient molecular detail of zona proteins toenable a new contraceptive approach. This strategy is based on activealloimmunization with a zona pellucida polypeptide which includes anamino acid sequence that is selected to display at least one epitope forbinding of an antibody that inhibits fertilization of an oocyte by asperm.

The complete nucleotide sequence of the mouse ZP3 messenger RNA and theamino acid sequence encoded thereby has been disclosed previously by thepresent inventor and associates (M. J. Ringuette et al., Dev. Biol.127:287-296 (1988), published Jun. 13, 1988, the entire contents ofwhich are hereby incorporated herein by reference). The completenucleotide sequence of the mouse ZP2 (Liang et al., Mol. Cell. Biol.10:1507-15 (1990)), the human ZP3 (Chamberlin et al., Proc. Natl. Acad.Sci. U.S.A. 87:6014-18 (1990)) and the human ZP2 (Liang et al., Mol.Cell. Biol. 10:1507-15 (1993)) messenger RNAs and the amino acidsequences encoded thereby have also been disclosed and the entirecontents of the published documents are hereby incorporated herein byreference.

The present inventor and associates have also reported (M. Chamberlin etal., 1987, abstract of a presentation at the twenty-seventh annualmeeting of the American Society for Cell Biology, St. Louis, Mo.,November 16-20, J. Cell Biol. 105, no. 4 part 2, 334A) that mousegenomic clones of the ZP3 gene and a human genomic DNA clone of the ZP3gene have been isolated by virtue of their homology to the previouslyisolated murine ZP3 cDNAS. However, this abstract does not disclosespecific nucleotide or amino acid sequences of any mouse or human DNAclone, nor does it even mention any concept of a contraceptive vaccine.Further, the mouse and human ZP2 cDNA sequences have not been disclosedpreviously.

Enabled by an oligonucleotide probe based on the short ZP3 cDNA sequencethat was published by the present inventor and associates (Ringuette etal., supra (1986)), and subsequent to publication of the complete mouseZP3 cDNA sequence (M. J. Ringuette et al., Dev. Biol. 127:287 (1988)),others have also reported isolation and sequences of genomic DNA clonesof a mouse ZP3 gene and the amino acid sequence encoded therein (R. A.Kinloch et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:6409-413 (1988)).This information was also used to isolate genomic DNA clones of hamsterZP3 and, by comparison with previously described mouse ZP3 genes, todeduce the amino acid sequence of the resultant polypeptide chain(Kinloch et al., Devel. Biol. 142:414-21 (1990)). Independently, othershave reported the isolation of a cDNA encoding rc55, a rabbit zonapellucida protein (Schwoebel et al., J. Biol. Chem. 266:7214-19 (1991)),that does not appear to be the homologue of either mouse ZP2 or ZP3.

Whereas the prior art on contraceptive vaccines based on zona pellucidaantigens has been and remains primarily focused on heteroimmunization,the present invention relates to contraceptive vaccines based on clonedzona pellucida genes and the strategy of alloimmunization withpolypeptides including defined amino acid sequences that are selectedfor displaying epitopes to contraceptive antibodies. The advantages ofthis approach include the ability to produce and utilize thoseimmunogens displaying the most effective B-cell epitopes for inhibitionof fertilization regardless of whether or not they happen to beconserved in several species. Further, this vaccine strategy minimizesthe likelihood of inducing antibodies with deleterious cross-reactivitywith epitopes on molecules other than zona pellucida polypeptides.Ultimately, by reducing in the vaccine the number of B-cell epitopesthat produce antibodies which, even though they bind to a zona pellucidaantigen, do not block conception, this invention focuses the immuneresponse to the vaccine on precisely those amino acids that are mostcritically situated to facilitate the contraceptive effect ofantibodies. Further, by focusing on those epitopes that are most usefulfor contraceptive purposes, the present invention minimizes potentialinterference with establishment of effective immunity to those criticalcontraceptive epitopes from extraneous epitopes that may beimmunodominant to those critical sites and, therefore, may prevent anadequate contraceptive antibody response to them. In addition, byfocusing on these epitopes, the potential for adverse immunologicalresponse due to the induction of autoimmune responses can be minimized(Rhim et al., J. Clin. Invest. 89:28-35 (1992)).

It is understood that, in the practice of the present invention,epitopes may be used which happen to be conserved in the zona pellucidaproteins of more than one species. However, in contrast to previousefforts to employ zona pellucida antigens in vaccines wherein the firstconcern has been to identify cross-reacting epitopes in heterologousantigens without initial regard for the functionality of such epitopesin inducing contraceptive antibodies, as described in some referencescited herein above, it will be appreciated that use of conservedepitopes in the instant invention is entirely incidental to the goal ofproviding epitopes that are effective for inducing contraceptiveantibodies in the particular target species intended for a givenvaccine.

Accordingly, the present invention relates to a contraceptive vaccinefor use in a mammalian female comprising a polypeptide which includes anamino acid sequence that is selected to display at least one epitope forbinding of an antibody that inhibits fertilization of an oocyte by asperm. This contraceptive antibody epitope is an epitope for which thereis a functional homolog displayed on a zona pellucida protein thatoriginates from the species in which the said vaccine is used. The zonapellucida protein displaying the functionally homologous epitopeadvantageously is either a ZP3 protein or a ZP2 protein or a ZP1protein.

In other words, both the amino acid sequence of a polypeptide of thisvaccine and a zona pellucida protein display epitopes which arefunctionally homologous in that they both are able to bind the sameantibody that inhibits fertilization of an oocyte by a sperm. The factthat this vaccine polypeptide and a zona pellucida protein both displayfunctionally homologous binding sites for the same antibody does notimply, however, that these binding sites are encoded by the same aminoacid sequence in each instance, i.e., the polypeptides displaying thetwo epitopes are not necessary structurally homologous at the level ofamino acid sequences encoding the epitopes.

By the phrase "originating from" it is meant that the zona pellucidaprotein is encoded in the genome of the species in which the saidvaccine is used.

It will be understood from the foregoing Background that thenomenclature of zona pellucida proteins comprising ZP1, ZP2 and ZP3 hasbeen defined in the mouse system and that other nomenclature or nonomenclature may be used in other mammalian systems. However, thepresent inventor has clearly demonstrated that the genes and mRNAS and,hence, the amino acid sequences of the major murine zona pellucidaproteins (for example, the ZP3 and ZP2 proteins of the mouse) areconserved throughout diverse mammalian species (see below). In light ofthis high degree of structural similarity, a high degree of functionalhomology is also to expected in terms of the ability of homologouspositions to serve as epitopes of contraceptive antibodies. Accordingly,the terms "ZP3 protein", "ZP2 protein", and "ZP1 protein" contemplatenot only the murine forms of these highly conserved zona pellucidaproteins, but also the homologous counterparts of any other mammalianspecies, regardless of any other terminology by which such otherproteins may be known in the art.

Contraceptive antibodies suitable for the practice of the presentinvention may be generated using zona pellucida antigens from naturalsources, according to various published procedures. Alternatively, suchantibodies may be produced advantageously by immunization with apolypeptide produced in a recombinant expression system comprising a DNAsegment of the present invention. Various methods for identifyingantibodies, including monoclonal antibodies, that inhibit thefertilization of an oocyte by a sperm have also been published (e.g.,East et al., Dev. Biol. 109:268 (1985)).

In the polypeptide of the vaccine of this invention, the amino acidsequence which displays an epitope for a contraceptive antibody mayinclude all or part of the same amino acid sequence responsible fordisplaying the functionally identical epitope on a zona pellucidaprotein. In some cases, a single epitope for binding a given antibodycomprises more than one contiguous amino acid sequence of a polypeptide(see discussion of "discontinuous epitope", below); accordingly, thepresent invention contemplates that the polypeptide of the vaccine mayinclude at least one amino acid sequence of a zona pellucida proteinthat displays a functionally homologous epitope.

An amino acid sequence displaying an epitope for an availablecontraceptive antibody may be selected from all the sequences in a zonapellucida protein using a known contraceptive antibody. For example, acontraceptive antibody may be used to isolate a peptide displaying itsepitope from a proteolytic digest of a zona pellucida protein by meansof affinity chromatography methods that are well known in the art.

Alternatively, a DNA sequence encoding an amino acid sequence whichdisplays an epitope for a contraceptive antibody may be isolated bystandard genetic engineering approaches. These involve screening ofclones of fragments of a gene for a zona pellucida protein for theability to express an amino acid sequence that binds the contraceptiveantibody. In addition, if sufficient zona proteins can be produced bystandard recombinant DNA techniques, it may be possible to determine the3-dimensional structure by standard biochemical techniques (e.g. nuclearmagnetic resonance, and X-ray diffraction).

Yet another way to identify an amino acid sequence that displays theepitope of a contraceptive antibody is to employ the well known strategyof chemical synthesis of every distinct peptide that could possiblydisplay an antibody epitope. For instance, technology is commerciallyavailable for the rapid synthesis and antibody reactivity testing of allpeptides of six amino acids that occur sequentially in the sequence of aprotein and overlap by one amino acid. In the practice of the presentinvention, the sequences to be synthesized are determined advantageouslyfrom the nucleotide sequence of a cloned gene for a zona pellucidaprotein.

In another embodiment of this aspect of the present invention, the aminoacid sequence that displays the epitope for a contraceptive antibody inthe vaccine may be some type of analog of the amino acid sequence forthat epitope on the zona pellucida protein.

One type of analog that this embodiment includes is a synthetic peptideknown as a "mimotope" by H. M. Geysen, the inventor of the technologyused to create such analogs, for which kits of materials are nowcommercially available. In a substantial number of cases, this syntheticepitope generation approach produces amino acid sequences that arefunctional analogs of known epitopes for a given antibody, and theseanalogs can induce other antibodies that recognize the same epitope asthe original selected antibody. These analog sequences, however, usuallydo not contain the amino acids in the natural amino acid sequence thatdisplays the selected epitope. Thus, this type of analog sequence mimicsa naturally occurring structure that displays an epitope, hence, theterm "mimotope". An important feature of this particular aspect of thisembodiment of the present invention is that it is not necessary toidentify the natural amino acid sequence displaying the epitope of thedesired contraceptive antibody; in fact, this method can produce smallpeptide analogs of natural epitopes comprising amino acids located indistinct positions of a protein that are separated by many amino acids(i.e., so-called "discontinuous epitopes" as opposed to those epitopesencoded by a single short continuous amino acid sequence).

In the term "analog", this aspect of the present invention alsocontemplates the application of well known principles of sequenceconservation during the evolution of protein families to identifyepitopes for contraceptive antibodies in a selected zona pellucidaprotein for which such antibodies are not yet available. If the aminoacid sequence of this zona pellucida protein is highly homologous tothat of related protein from another species, and if epitopes for suchcontraceptive antibodies have been defined in the sequence of thislatter protein, then the general structural homology between the twoproteins may be used to indicate those sequences in the selected proteinthat display epitopes for contraceptive antibodies that are analogous tothose known for the second protein.

In other words, when two short, distinct amino acid sequences are knownto occupy the same position in two proteins of substantially homologousstructure (i.e., overall amino acid sequence and, consequently,three-dimensional conformation), then if one of the two sequencesdisplays an epitope for an antibody with a particular biological effect,the other sequence has a high probability of displaying epitopes forother antibodies with the same biological effect. According to thisaspect of this invention, a known epitope for a contraceptive antibodyis embodied by an amino acid sequence identified in a mouse ZP3 proteinby screening cloned fragments of a cloned DNA for expression of suitableepitopes, and one analog of this amino acid sequence is embodied by thesequence of amino acids that occupies the homologous position in thehuman ZP3 protein. A second epitope for a known contraceptive antibodyis embodied by an amino acid sequence identified in a mouse ZP2 proteinas above and one analog of this amino acid sequence is embodied by thesequence of amino acids which occupies the homologous position in thehuman ZP3 protein. This human analog of a mouse ZP3 or ZP2 epitope(which also may be considered to be a "homologue" of that epitope), isto be incorporated into a vaccine for use in human beings, of course,according to the alloimmunization aspect of the present invention.

It is understood that chemically synthesized peptides may be usedadvantageously as polypeptides of the present invention, especiallysince the synthesis of such peptides comprising 30 to 50 or even moreamino acids can now be achieved on scales sufficient for vaccinepurposes (in batches of 1 gram or more, for example). One such syntheticpeptide is embodied by a mouse ZP3 peptide and a mouse ZP2 peptide thatare described below.

It should be particularly noted that the polypeptides of the presentinvention do not include idiotypic antibodies or large fragments of suchantibodies, since the disadvantages of using such polypeptides topresent epitopes of zona pellucida proteins has been discussed above inthe Background in regard to prior art on such antibodies. However, thepresent invention does contemplate smaller polypeptides comprisingmainly those amino acid sequences of such idiotypic antibodies thatactually comprise the analog of the original zona pellucida proteinepitope.

Further, as will be appreciated from the Background discussion ofimmunogenicity of polypeptides, the immunogenicity of polypeptides orpeptides of the present invention in terms of raising higher titers ofcontraceptive antibodies with greater affinities for their epitopes,particularly such immunogenicity of small (synthetic) peptides, may beenhanced advantageously by covalent coupling to another polypeptide orpeptide, especially to another amino acid sequence displaying a T-cellepitope. In addition, it will be appreciated that, as is customary forvaccines, the polypeptides of the present invention will be delivered ina pharmacologically acceptable vehicle. Vaccines of the presentinvention may also advantageously comprise effective amounts ofimmunological adjuvants that are known to enhance the immune response toimmunogens in general, particularly adjuvants that enhance theimmunogenicity of small synthetic peptides.

In another aspect, the present invention further relates to certain DNAsegments that encode mouse ZP3 or ZP2 proteins and human ZP3 or humanZP2 proteins. This invention also relates to cultures of recombinantcells containing a DNA segment of this invention and to methods for thesynthesis and isolation of polypeptides and peptides of this invention.

The present invention also relates to recombinant DNA moleculescomprising a DNA segment of this invention and a vector. A number ofvectors may be utilized such as, for example, the vaccinia virus.

In particular, the present invention includes a contraceptive vaccinefor use in a mammalian female comprising a polypeptide which consistsessentially of the mouse zona pellucida 3 (ZP3) amino acid sequenceCys-Ser-Asn-Ser-Ser-Ser-Ser-Gln-Phe-Gln-Ile-His-Gly-Pro-Arg-Gln SEQ IDNO:12 or a homologous mammalian amino acid sequence derived from ahomologous region of a ZP3 protein. The mammalian amino acid sequence isincluded in a zona pellucida protein originating from the species inwhich the vaccine is used. The vaccine also includes a pharmacologicallyacceptable vehicle. It must be noted that portions of the sequence mayalso be utilized in the vaccine.

The homologous mammalian amino acid sequence in the vaccine may be, forexample, Cys-Gly-Thr-Pro-Ser-His-Ser-Arg-Arg-Gln-Pro-His-Val-Met-Ser-GlnSEQ ID NO:11. This sequence is derived from a human ZP3 protein. Itshould be noted that portions of the 16 amino acid sequence may beutilized in the vaccine.

Additionally, the present invention relates to a contraceptive vaccinefor use in a mammalian female comprising a polypeptide which consistsessentially of the mouse zona pellucida 2 (ZP2) amino acid sequenceIle-Arg-Val-Gly-Asp-Thr-Thr-Thr-Asp-Val-Arg-Tyr-Lys-Asp-Asp-Met SEQ IDNO:10 or a homologous mammalian amino acid sequence derived from ahomologous region of a ZP2 protein. The mammalian amino acid sequence isincluded in a zona pellucida protein originating from the species inwhich the vaccine is used. The vaccine also includes a pharmacologicallyacceptable vehicle. Portions of the 16 amino acid sequence may beutilized in the vaccine.

The homologous mammalian amino acid sequence referred to above may be,for example,Ile-Arg-Val-Met-Asn-Asn-Ser-Ala-Ala-Leu-Arg-His-Gly-Ala-Val-Met SEQ IDNO:9 and is derived from a human ZP2 protein. It should be noted thatportions of the 16 amino acid sequence may also be utilized in thevaccine.

Each of the above-vaccines may include an effective amount of anadjuvant. Furthermore, the mammalian female may be a cat, a dog, a pig,a cow, or a woman. It is important to note that the polypeptide isderived from the same species to which it is administered in vaccineform.

The present invention also relates to a contraceptive vaccine comprisinga polypeptide which consists essentially of a synthetic peptidecorresponding to the mouse zona pellucida (ZP3) amino acid sequenceCys-Ser-Asn-Ser-Ser-Ser-Ser-Gln-Phe-Gln-Ile-His-Gly-Pro-Arg-Gln or asynthetic peptide corresponding to a homologous mammalian amino acidsequence derived from a homologous region of a ZP3 protein, for bindingof an antibody that inhibits fertilization of an egg by a sperm. Themammalian amino acid sequence, as noted above, is included in a zonapellucida protein originating from the species in which said vaccine isused. The vaccine may further comprise a pharmacologically acceptablevehicle. Portions of the 16 amino acid sequence may also be used.

The homologous mammalian amino acid sequence may be, for example,Cys-Gly-Thr-Pro-Ser-His-Ser-Arg-Arg-Gln-Pro-His-Val-Met-Ser-Gln and isderived from a human ZP3 protein. Portions of the homologous sequencemay be utilized in the vaccine.

The present invention also relates to a contraceptive vaccine comprisinga polypeptide which consists essentially of a synthetic peptidecorresponding to the mouse zona pellucida (ZP2) amino acid sequenceIle-Arg-Val-Gly-Asp-Thr-Thr-Thr-Asp-Val-Arg-Tyr-Lys-Asp-Asp-Met or asynthetic peptide corresponding to a homologous mammalian amino acidsequence derived from a homologous region of a ZP2 protein, for bindingof an antibody that inhibits fertilization of an egg by a sperm. Themammalian amino acid sequence is included in a zona pellucida proteinoriginating from the species in which the vaccine is used. The vaccinemay further comprise a pharmacologically acceptable vehicle. It shouldbe noted that portions of the sequence shown above may be utilized inthe vaccine.

The homologous mammalian amino acid sequence may be, for example,Ile-Arg-Val-Met-Asn-Asn-Ser-Ala-Ala-Leu-Arg-His-Gly-Ala-Val-Met which isderived from a human ZP2 protein. Portions of this sequence may beutilized in the vaccine.

Additionally, the present invention also includes a DNA segment encodingthe mouse ZP3 protein or a portion thereof, a DNA segment encoding themouse ZP2 protein or a portion thereof, a DNA segment encoding the humanZP3 protein or a portion thereof, and a DNA segment encoding the humanZP2 protein or a portion thereof.

The invention also encompasses a recombinant DNA molecule comprising aDNA segment encoding the human ZP3 or human ZP2 protein, or a portion ofeach protein, and a vector. Additionally, the invention includescultures of host cells transformed or transfected with the recombinantDNA molecules or constructs.

The invention also includes a method of producing at least a portion ofa human ZP3 or human ZP2 protein comprising culturing the above-cellsunder conditions such that the protein is produced and isolating saidprotein from culture media or from the cells.

The invention also includes an antibody specific for a protein havingthe amino acid sequence of the human ZP3 or ZP2 protein or a portionthereof. This antibody inhibits fertilization of a human oocyte by asperm.

Furthermore, the invention also includes the purified proteins encodedby the DNA segments referred to above. All U.S. patents and publicationsreferred to herein are hereby incorporated by reference.

The present invention may be understood more readily by reference to thefollowing detailed description of specific embodiments and the Examplesand Figures included therein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Nucleic Acid Sequence of Mouse ZP3 cDNA SEQ ID NO:4 and DeducedAmino Acid Sequence SEQ ID NO:4 of Mouse ZP3 Protein.

The nucleic acid sequence of near-full-length cDNAs and exon 1 of theZp-3 gene were used to deduce the structure of the ZP3 mRNA andresultant protein. The initiation and termination codons are boxed, andthe polyadenylation signal is overlined. The single 1272-nt open readingframe is translated into a 424 amino acid ZP3 protein in line 2. Theproposed 22-amino acid signal peptide is indicated by a wavy line, andthe arrow points to the proposed signal peptidase cut site. Amino acidsequences which were experimentally determined by isolation and directsequencing of an internal ZP3 peptide are underlined with a dashed line.The 6 potential N-linked glycosylation sites (Asn-X-Thr/Ser) areunderlined with a solid line (Ringuette et al., Dev. Biol. 127:287-95(1988)).

FIG. 2: Nucleic Acid Sequence of Human ZP3 cDNA SEQ ID NO:3 and DeducedAmino Acid Sequence of Human ZP3 Protein Compared to the Amino AcidSequence of Mouse ZP3 Protein SEQ ID NO:8.

The first line is the nucleic acid sequence of human ZP3 mRNA containing1289 nt determined from human cDNA and genomic sequences (Chamberlin etal., Proc. Natl. Acad. Sci. USA 87:6014-18 (1990)). The initiation andstop codons are boxed, and the polyadenylation signal overlined. Thesingle 1272-nt open reading frame is translated into a 424-amino acidpeptide in the second line and aligned in the third line with the 424amino acids of mouse ZP3 protein (Ringuette et al., Dev. Biol.127:287-95 (1988)).

Identical amino acid residues in mouse and human ZP3 are shaded;conserved changes (Dayhoff et al., Proc. Natl. Acad. Sci. USA 76:2170-74(1979)) are enclosed in boxes with dotted lines. The putative 22-aminoacid signal peptide is indicated by a wavy line, and the arrow points tothe proposed signal peptidase cut site. The four potential N-linkedglycosylation sites Asn-Xaa-(Thr or Ser)! of human ZP3 are bracketedfrom above, and the six potential sites of mouse ZP3 are bracketed frombelow. Three of the sites are conserved between the two species.

FIG. 3: Nucleic Acid Sequence of Mouse ZP2 cDNA SEQ ID NO:2 and DeducedAmino Acid Sequence of Mouse ZP2 Protein SEQ ID NO:6.

The nucleic acid sequence of the near-full-length cDNAs and exon 1 ofthe Zp-2 gene were used to deduce the structure of the ZP2 mRNA andresultant protein. The initiation and termination codons are boxed, andthe polyadenylation signal is overlined. The single 2139 nucleotide openreading frame is translated into a 713 amino acid protein in line 2. The34-amino acid signal peptide is indicated by a wavy line, and the arrowpoints to the signal peptidase cut site. Amino acid sequences which wereexperimentally determined by isolation and direct sequencing of anN-terminal and an internal ZP2 peptide are underlined with a dashedline. The 7 potential N-linked glycosylation sites (Asn-X-Thr/Ser) areunderlined with a solid line (Liang et al., Mol. Cell. Biol. 10:1507-15(1990)).

FIG. 4: Nucleic Acid Sequence of Human ZP2 cDNA SEQ ID NO:1 and DeducedAmino Acid Sequence of Human ZP2 Protein SEQ ID NO:5 Compared to theAmino Acid Sequence of Mouse ZP2 Protein SEQ ID NO:6.

The nucleic acid sequence of the near-full-length cDNAs and exon 1 ofthe ZP2 gene were used to deduce the structure of the ZP2 mRNA andresultant protein (Liang et al., Dev. Biol. 156:399-408 (1993)). Theinitiation and termination codons are boxed, and the polyadenylationsignal is overlined. The single 2235 nucleotide open reading frame istranslated into a 745 amino acid protein in line 2 and aligned in line 3with the 713 amino acid mouse ZP2 (Liang et al., Mol. Cell. Biol.10:1507-15 (1990)). The putative 38-amino acid signal peptide isunderlined, and the arrow points to the predicted signal peptidase cutsite. Identical amino acid residues between human and mouse ZP2 areshaded; conserved changes (Dayhoff et al., Proc. Natl. Acad. Sci. USA76:2170-74(1979)) are enclosed in boxes with dotted lines. The potentialN-linked glycosylation sites (Asn-X-Thr/Ser) are marked in boldbrackets.

FIG. 5: Exon Maps of Mouse and Human ZP2 Genes and of Mouse and HumanZP3 Genes.

Dark vertical bars represent the exons. The double-headed arrowrepresents 2kbp of genomic DNA. Mouse Zp-2 is a single copy genecontaining 18 exons which span 12.1-kbp (Liang et al., Mol. Cell. Biol.10:1507-15 (1990)). Human ZP2 contains 19 exons, spanning 14-kbp (Lianget al., supra (1993)). Mouse Zp-3 is a single copy gene spanning 8.6-kbpand containing 8 exons (Chamberlin et al., Dev. Biol. 131:207-14(1989)). Human ZP3 spans 18.3-kbp and contains 8 exons (Chamberlin etal., Proc. Natl. Acad. Sci. USA 87: 6014-18 (1990)). The size anddistribution of the exons is well conserved between the two species forboth ZP2 and for ZP3 genes.

FIG. 6: Comparison of the Secondary Structures of Mouse and Human ZP2Proteins and of Mouse and Human ZP3 Proteins.

The hydropathicity of the 713 amino acid mouse ZP2 (Liang et al., Mol.Cell. Biol. 10: 1507-15 (1990) and 745 amino acid human ZP2 (Liang etal., Dev. Biol. 156: 399-408 (1993)), determined by the Kyte andDoolittle algorithm (Kyte et al., J. Mol. Biol. 157: 105-32 (1982)),indicates the overall similarity of the two proteins. Both have majorhydropathic peaks in their signal peptides and near their carboxyltermini. The hydropathicity of the 424 amino acid mouse ZP3 (Ringuetteet al., Dev. Biol. 127: 287-295 (1988)) and 424 amino acid human ZP3(Chamberlin et al., 87: 6014-18 (1990)), determined by the Kyte andDoolittle algorithm (Kyte et al., supra), indicates the overallsimilarity of these two proteins. Both have major hydropathic peaks intheir signal peptides and near their carboxyl termini.

FIG. 7: The Definition of a Potential Zona Pellucida Peptide for Use asa Contraceptive Vaccine by Screening a ZP3 Epitope Library with aMonoclonal Antibody Specific to ZP3.

(A) Schematic representation of the 1317 nucleotide ZP3 mRNA. The single1272-nt open reading frame is indicated by an open bar. The lines belowthe mRNA represent eight positive cDNA clones isolated from the ZP3epitope library by the monoclonal antibody to ZP3. The clones arealigned on the ZP3 cDNA and the hatched bar indicates the sequencecommon to all positive clones.

(B) The DNA sequence of the overlapping regions among the eight positiveclones and the corresponding amino acid sequence (bold) are shown. Theone additional COOH-terminal and eight additional NH₂ -terminal aminoacids shown flanking the epitope were included in the peptide used forimmunization.

(C) Hydrophilicity of the deduced 424-amino acid ZP3 protein was plottedwith a seven-residue moving average. Horizontal filled-in bars beneaththe hydrophilicity indicate amphipathic α helical segments predicted byan 11-residue moving average. The speckled vertical bar represents the16-amino acid peptide shown in 7B) that was used to immunizeexperimental animals (Millar et al., Science 246: 935-38 (1989)).

FIG. 8. Localization of Two Monoclonal Antibody Binding Sites on MouseZP2 and ZP3.

(A) Hydrophilicity of the 713-amino acid ZP2 protein plotted with aseven-residue moving average. Horizontal filled-in bars beneath thehydrophilicity plot indicate amphipathic α helical segments predicted byan 11-residue moving average. The speckled vertical bar represents the16-amino acid peptide that is the binding site of a monoclonal antibodyspecific to mouse ZP2.

(B) Hydrophilicity of the 424-amino acid ZP3 protein plotted with aseven-residue moving average. Horizontal filled-in bars beneath thehydrophilicity plot indicate amphipathic α helical segments predicted byan 11-residue moving average. The speckled vertical bar represents the7-amino acid peptide that is the binding site of a monoclonal antibodyspecific to mouse ZP3.

FIG. 9: Alignment of the Mouse ZP3 SEQ ID NO:12 and ZP2 SEQ ID NO:10Epitopes with the Homologous Portions of the Human ZP2 SEQ ID NO:9 andZP3 Proteins SEQ ID NO:11.

(A) Mouse ZP3 amino acids 328-343 aligned with human ZP3 amino acids327-342 (see FIG. 2).

(B) Mouse ZP2 amino acids 114-129 aligned with human ZP2 amino acids118-133 (see FIG. 4).

DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention relates in part to DNA segments having sequencesthat encode mouse and human ZP3 and ZP2 proteins. An embodiment of thisaspect of the invention includes cDNA and genomic clones that encode atleast a portion of the complete nucleotide sequence of the mouse ZP3mRNA and the protein encoded thereby which has been described by thepresent inventor in Example 1, below, and has been published (Ringuetteet al., Dev. Biol. 127:287-95 (1988); Chamberlin et al., Dev. Biol.131:207-14 (1989)).

A second embodiment of this aspect of the invention includes cDNA andgenomic clones that encode at least a portion of the complete nucleotidesequence of the mouse ZP2 mRNA and the protein encoded thereby which hasbeen described by the present inventor in Example 1, below, and has beenpublished (Liang et al., Mol. Cell. Biol. 10:1507-15 (1990)).

A third embodiment of this aspect of the invention includes cDNA andgenomic clones that encode at least a portion of the complete nucleotidesequence of the human ZP3 mRNA and the protein encoded thereby which hasbeen described by the present inventor in Example 1, below, and has beenpublished (Chamberlin et al., Proc. Natl. Acad. Sci. USA 87:6014-18(1990)).

A fourth embodiment of this aspect of the invention include cDNA andgenomic clones that encode at least a portion of the complete nucleotidesequence of the human ZP2 mRNA and the protein encoded thereby which hasbeen described by the present inventor in Example 1, below, and has beenpublished (Liang et al., Dev. Biol. 156:399-408 (1993)).

A summary of this information follows:

Genomic Organization and Conservation of the Zona Pellucida Gene: MouseZp-2 and Zp-3 are each present in a single copy in the mouse genome andare present on different chromosomes. Zp-2 is located on chromosome 7,11.3±3.2 cM distal to the Tyr locus and Zp-3 is located on chromosome 5,9.2±2.9 cM distal to the Gus locus (Lunsford et al., Genomics 6:184-87(1990)). Mouse Zp-2 contains 18 exons that range in size from 45 bp to190 bp separated by 17 introns (81 bp to 1490 bp) and spans 12.1-kbp ofDNA (FIG. 5)(Liang et al., supra (1990)). The 8.6-kbp long mouse Zp-3gene contains 8 exons ranging in size from 92 bp to 338 bp and hasintrons whose lengths are between 125 bp and 2320 bp (FIG. 5)(Chamberlinet al., Dev. Biol. 131:207-14 (1989)). The intron-exon boundaries ofboth genes contain consensus splice donor/acceptor sites (Breathnach etal., Annu. Rev. Biochem. 50:349-83 (1981)).

The genes encoding ZP2 and ZP3 are conserved among mammals. Takingcross-hybridization of nucleic acid sequences as a criteria, the degreeof conservation of Zp-3 is variable with pig and rabbit being lessrelated to mouse than rat, dog, cow and human zona genes (Ringuette etal., Proc. Natl. Acad. Sci. U.S.A. 83:4341-45 (1986)). The human homologof Zp-2 and Zp-3 have been isolated using standard genetic engineeringapproaches well known in the art by virtue of their homology to thepreviously isolated murine ZP2 and ZP3 cDNAs. The human ZP2 gene iscomposed of 19 exons (FIG. 5) whose nucleic acid sequence is 70% thesame and encodes a 745 amino acid protein that is 60% identical to thatof its mouse counterpart (FIG. 4)(Liang et al., Dev. Biol. 156:399-408(1993)). The mouse and human ZP3 genes each contain 8 exons. The codingsequence of the mouse and human genes are 74% the same and each encodesa 424 amino acid peptide that is 67% identical (FIG. 2)(Chamberlin etal., Proc. Natl. Acad. Sci. U.S.A. 87:6014-18 (1990)).

ZP2 mRNA and Protein: The structure of mouse ZP2 was deduced fromnear-full-length cDNA clones and genomic clones containing exon 1. TheZP2 mRNA is 2201 nt long with very short 5' (30 nt) and 3' (32 nt)untranslated regions (FIG. 3). A transcript of approximately 2.4-kbp isobserved by Northern blot analysis of oocyte RNA suggesting that ZP2mRNA contains a poly(A) tail of approximately 200 nt (Liang et al., Mol.Cell. Biol. 10:1507-15 (1990)). ZP2 mRNA has a single open reading frameof 2139 nt initiated at an ATG within the ANNATG motif associated withvertebrate initiator codons (Kozak, Cell 44:283-93 (1986); Cavener,Nucleic Acids Res. 15:1353-61 (1986)). The open reading frame encodes apolypeptide of 713 amino acids with a molecular weight of 80,217daltons, the amino acid composition of which is 10.8% acidic, 9.5%basic, 10.2% aromatic and 34.8% hydrophobic.

The first 34 amino acids of the deduced polypeptide are absent from theN-terminal amino acid sequence obtained from SDS-PAGE purified ZP2protein and presumably represent a signal peptide. The amino acids atthe -1 and -3 position from the presumptive signal peptidase cleavagesite are Ser and Asn, respectively. The positions of these two aminoacids are similar to other eukaryotic signal peptidase cleavage sites(Perlman et al., J. Mol. Biol. 167:391-409 (1983)) and are in accordancewith the (-3, -1) rule of signal peptidase cleavage sites proposed byvon Heijne (Von Heijne, J. Mol. Biol. 184:99-105 (1985); Von Heijne,Nucleic Acids Res. 14:4683-90 (1986)). The resultant core polypeptidesecreted into the extracellular matrix would have a molecular weight of76,373 daltons. The ZP2 amino acid sequence contains seven possibleN-linked glycosylation sites (Asn-X-Ser/Thr), and more than 100potential O-linked glycosylation sites (Liang et al., supra (1990)).

The human and mouse ZP2 mRNAs and proteins are well conserved. The humanZP2 mRNA contains an open reading frame of 2235 nt that can code for apolypeptide of 82,356 daltons containing 745 amino acids (10.2% acidic,11.5% basic, 9.4% aromatic and 50.3% hydrophobic). Human and mouse ZP2amino acid sequences are 60.7% identical. Examination of human ZP2protein revealed a potential signal peptidase cleavage site whichcontains amino acids at the -1 and -3 positions that are in accordancewith the (-3, -1) rule proposed by von Heijne (Von Heijne, J. Mol. Biol.184:99-105 (1985); Von Heijne, Nucleic Acids Res. 14:4683-90 (1986)).Cleavage at the presumptive signal peptidase site would give rise to asignal sequence of 38 amino acids (4 residues longer than mouse ZP2) anda resultant protein with a predicted molecular mass of 78,200 daltons.The deduced polypeptide chain contains six potential N-linkedglycosylation sites (Asn-X-Ser/Thr), four of which are conserved in themouse ZP2 polypeptide (FIG. 4). The predicted hydropathicity of thehuman and mouse ZP2 proteins are quite similar, reflecting both aminoacid identity and conservative amino acid substitutions (FIG. 6). Theconservation of all 20 cysteine residues in the mature human and mouseproteins suggests that at least some of these residues participate indisulfide bonds important for tertiary structure. An additional exonfound in human ZP2 (FIG. 5) encodes a 28 amino acid hydrophilic regioll(residues 671-698) near the carboxyl terminus.

ZP3 mRNA and Protein: Primer extension studies and S1 nucleaseprotection assays were used to define the 5' terminus of the mouse ZP3mRNA. Similar to ZP2 mRNA, the 1317 nt ZP3 mRNA has short 5' (29 nt) and3' (16 nt) untranslated regions (FIG. 1). The latter is so abbreviatedthat the TAA termination codon is embedded within the consensus AATAAApolyadenylation signal (Ringuette et al., Dev. Biol. 127:287-95 (1988)).It is not clear what the role, if any, that these short untranslatedregions play in gene expression nor whether they are important forprocessing ZP2 and ZP3 transcripts. This short untranslated region is acharacteristic of both ZP2 (mouse and human) and ZP3 (mouse and human)mRNAs. The mouse ZP3 mRNA in oocytes is 1.5-kb, indicating that it has apoly(A) tail of 200 nt, and is indistinguishable in size from that ofrat and rabbit (Ringuette et al., supra (1988)). Taken together, thesedata suggest that the overall structure of ZP3 mRNA is conserved amongmammals.

The polypeptide deduced from the single open reading frame of mouse ZP3mRNA is 46,307 daltons consisting of 424 amino acids (9% acidic, 7.3%basic, 7.5% aromatic and 31.4% hydrophobic). The N-terminal amino acidof the secreted glycoprotein is blocked to Edman degradation, but usingthe sliding window/matrix scoring method of von Heijne (Von Heijne,supra (1985); Von Heijne, supra (1986)), a potential signal peptide of22 amino acid has been identified (Ringuette et al., supra (1988)). Theresultant secreted protein would have a molecular weight of 43,943daltons, consistent with the reported 44,000 dalton ZP3 core protein(Bleil et al., Dev. Biol. 76:185-202 (1983)).

The human and mouse ZP3 mRNAs and proteins are well conserved. The humanZP3 mRNA contains an open reading frame of 1272 nt that can code for apolypeptide of 47,032 daltons containing 424 amino acids (12% acidic, 8%basic, 7% aromatic and 32% hydrophobic). Human and mouse ZP3 amino acidsequences are 67% identical. Examination of human ZP3 protein revealed apotential signal peptidase cleavage site which contains amino acids atthe -1 and -3 positions that are in accordance with the (-3, -1) ruleproposed by von Heijne (Von Heijne, supra (1985); Von Heijne, supra(1986)). Cleavage at the presumptive signal peptidase site would giverise to a signal sequence of 22 amino acids and a resultant protein witha predicted molecular mass of 44,399 daltons. The deduced polypeptidechain contains four potential N-linked glycosylation sites(Asn-X-Ser/Thr), three of which are conserved in the mouse ZP3polypeptide (FIG. 2). The predicted hydropathicity of the human andmouse ZP3 proteins are quite similar, reflecting both amino acididentity and conservative amino acid substitutions (FIG. 6). Theconservation of all 13 cysteine residues in the mature human and mouseproteins suggests that at least some of these residues participate indisulfide bonds important for tertiary structure.

Conservation of Zona Protein Structure: The data in FIGS. 2, 4, 5, and 6clearly show the high homology of the mouse and human ZP3 and ZP2sequences, as would be expected from the extensive nucleic acidhybridization observed between mouse ZP3 cDNA and genomic DNAs from avariety of other mammalian species (see Example 2). From this structuralhomology data, and further standard analyses thereof (e.g., predictionsof secondary structure, hydropathicity, or surface accessibility), itwould be apparent to one of average skill in the art of proteinstructure and immunology that the mouse and human ZP3 proteins must alsoexhibit throughout their entire sequences, an extremely high level offunctional homology with respect to locations that are able to induceand bind contraceptive antibodies. Thus, although epitopes forcontraceptive antibodies on each protein may comprise short amino acidsequences which are not precisely conserved between the two proteins,the human sequences corresponding to such epitopes on the mouse proteinare also expected to induce functionally homologous antibodies, eventhough the mouse and human antibodies might only recognize theirrespective alloantigens.

It will be obvious, of course, to one of ordinary skill in the art ofgenetic engineering, that the above ZP3 and ZP2 sequences may varyslightly (i.e., be mutated) from one inbred mouse strain to another, orfrom one individual in an outbred population (e.g., one human being) toanother, without materially affecting the immunological character of thecorresponding zona pellucida protein and, therefore, without departingfrom the scope of the DNAs of the present invention as conveyed, forexample, by the use of the terms "the mouse ZP3 protein" or "the humanZP3 protein" or "the mouse ZP2 protein" or "the human ZP2 protein".

The DNA segments of the present invention variously enable developmentof different embodiments of the main aspect of the present invention,namely contraceptive vaccines for use in a mammalian female comprising apolypeptide which includes an amino acid sequence that is selected todisplay at least one epitope for binding of an antibody that inhibitsfertilization of an oocyte by a sperm. This contraceptive antibodyepitope is an epitope for which there is a functional homolog displayedon a zona pellucida protein that originates from the species in whichthe vaccine is used. The zona pellucida protein displaying thefunctionally homologous epitope advantageously is either a ZP3 proteinor a ZP2 protein or a ZP1 protein.

A principal embodiment of this aspect of this invention are twocontraceptive antibody epitopes that are displayed either on the mouseZP3 or the mouse ZP2 protein. Synthetic peptides containing either ofthese epitopes, when coupled to a carrier protein, for example, KLH,will elicit antibodies after alloimmunizations that react with the zonapellucida. FIG. 7 outlines the definition of the mouse ZP3 epitope for acontraceptive antibody, which is described in further detail in Example3, below. A similar strategy was employed to define the mouse ZP2epitope for a second contraceptive antibody. The ZP2 and ZP3 epitopesalong with their human homologues are shown in FIG. 9.

In brief, a cDNA encoding ZP3 was randomly fragmented and 200-500 bpfragments were cloned into the expression vector λgt11. This epitopelibrary was screened with the aforementioned anti-ZP3 contraceptivemonoclonal antibody and the positive clones were used to map a sevenamino acid epitope (amino acids 336-342) on mouse ZP3 recognized by theantibody. The homologous region on human ZP3 is contained in amino acids335-341.

In a similar fashion, a cDNA encoding ZP2 was randomly fragmented tocreate a second epitope library which was screened with theaforementioned anti-ZP2 contraceptive monoclonal antibody. Positiveclones were used to define a 16 amino acid epitope (amino acids 114-129)on mouse ZP2 recognized by the antibody. The homologous region on humanZP2 is contained in amino acids 118-133.

Of course, it must be noted that a shorter portion of the 7 amino acidsequence that displays the ZP3 epitope or the 16 amino acid sequencethat displays the ZP2 epitope might also be an effective peptide forpurposes of the present invention. Furthermore, certain analogues (e.g.,those sequences with ends that are chemically modified to neutralizecharges) might provide effective peptides for the practice of thepresent invention.

Female mice were immunized with a synthetic peptide containing the ZP3epitope, as described in Example 4, and the resultant circulatinganti-ZP3 antibodies bound to the oocytes of immunized animals producinglong-lasting contraception. As evidence that the effectiveness ofalloimmunization with a zona pellucida peptide is not limited to the ZP3protein, additional female mice were immunized with a synthetic peptidecontaining the ZP2 epitope, as described in Example 4. This vaccinationalso elicited antibodies that bound to the zona pellucida proteins.

The reversibility of the contraceptive effect, described in Example 4,can be accounted for by resting oocytes entering into the growth phaseand synthesizing a zona pellucida in the presence of low-levels ofcirculating anti-zona antibodies which appear to decline afterimmunization with the vaccine is terminated. When ovulated, theseoocytes would be coated lightly, if at all, with anti-zona antibodiesand would, therefore, be capable of being fertilized.

Studies have demonstrated that repeated immunization of female mice witha mouse ZP3 peptide-KLH conjugate results in long-term infertility inthe majority of cases. The production of anti-zona pellucida antibodiesoccurs despite the fact that the zona peptide is a self antigen(alloantigen). Immune tolerance has been postulated to occur in theneonatal period of development and involves both the functionalinactivation of B cells and the deletion of T cells which recognize selfantigens. The lack of detectable zona proteins in the ovary until 2-3days after birth, or their inaccessibility to the developing immunesystem, may account for the continued presence of lymphocytes capable ofrecognizing at least one ZP3 epitope.

In regard to the eventual reversibility of the contraceptiveimmunization, it is curious that having mounted an immunologicalresponse against the ZP3 peptide-KLH conjugate, the immune system doesnot continue to be stimulated by the endogenous ZP3 protein. Thefollowing hypotheses may account for this phenomenon in whole or inpart, and, therefore, aid in understanding the present invention; butthese theoretical explanations should not be construed to limit thescope of the present invention in any way. Nevertheless, it may bespeculated that one or more of the following may be involved in thereversibility of the contraceptive immunization: 1) The localization ofthe zona proteins uniquely to the ovary coupled with the lack ofcapillaries beyond the basement membrane surrounding the follicles, mayphysically preclude lymphocytes from interacting with and beingstimulated by the zona pellucida; 2) The 16 amino acid ZP3 peptideportion of the immunogen provides a B-cell epitope but may not containT-cell epitopes (which may, instead, be provided by the KLH moiety) tostimulate helper T-cell functions. Thus, the endogenous ZP3 protein,although containing the same ZP3 peptide, would not contain the T-cellepitopes of the carrier protein that, according to this hypothesis,could be important for mounting an anti-ZP3 peptide response; 3) Theovary may be part of an immunologically protected region and mechanismsthat suppress the immunological rejection of the embryo (which containspaternal and, thus, foreign antigens) also function in the ovary.

It is particularly important to note that immunization with the ZP3peptide vaccine did not result in either structural or functionalabnormalities of the mouse ovary (viz normal histology and the abilityof vaccinated females to subsequently have litters). In this regard, ofcourse, the use of a synthetic ZP3 peptide as a vaccine precludes anypossible minor contamination with other ovarian immunogens. In addition,the physical barrier of the follicular basement membrane and theextra-cellular site of the zona protein may contribute to the absence ofan immunocytotoxic response in the ovary. It should be noted that anearby, partially overlapping T cell epitope is able to elicit aninflammatory response in some but not other inbred strains of mice knownto be susceptible to autoimmune oophoritis (Rhim et al., J. Clin.Invest. 89: 28-35 (1992)). The potential to elicit an antibody responsein the absence of an ovarian inflammatory response may be an additionaladvantage of this invention (Millar et al., Targeting of zona pellucidafor immunocontraception, in Immunology of Reproduction, Naz, R. K, (ed)pp. 293-313 (1993)).

The mouse ZP3 epitope recognized by the monoclonal antibody used todevelop this vaccine is not detected immunologically in hamster, guineapig, cat or dog ovaries. Thus, this ZP3 peptide would not be expected toact as a contraceptive in other mammalian species, including humanbeings, although the ability of this antibody to bind to the human ZP3protein has not been tested. However, the strategy of the presentinvention of using vaccination with "self" zona peptides can be appliedto other species by taking advantage of the highly conserved nature ofthe zona genes among mammals. As noted above, the human homologues ofthe mouse ZP3 and ZP2 genes have been characterized, and the high degreeof structural homology is one indication of comparable functionalhomology in relation to epitopes for contraceptive antibodies.

Accordingly, using the deduced primary amino acid sequence of the humanZP3 and ZP2 proteins, by the practice of the present invention withoutundue experimentation, it is believed that one of ordinary skill in theart of polypeptide structure and immunology can identify in the human orother mammalian ZP3 and ZP2 proteins the region homologous to the mouseZP3 and ZP2 peptides described herein. Alternatively, one of such skillmay use computer algorithms to predict additional epitopes which may bepotential immunogens (T. P. Hopp and K. R. Woods, Proc. Natl. Acad. Sci.USA 78:3824 (1981); H. Maragalit, et al. J. Immunol. 138:2213 (1987); J.B. Rothbard and W. R. Taylor, EMBO J. 7:93 (1988)), or test a largearray of peptides representative of the polypeptide chain for epitopesof contraceptive antibodies using well known methods (H. M. Geysen etal. Proc. Natl. Acad. Sci. USA 81:3998 (1984); R. A. Houghten, Proc.Natl. Acad. Sci. USA 82:5131 (1985); H. M. Geysen, et al. Science235:1184 (1987); E. Norrby, et al. Proc. Natl. Acad. Sci. USA 84:6572(1987)).

Further, as noted previously, one skilled in the art of syntheticpeptide vaccines can also develop "mimotopes" of epitopes to availablecontraceptive antibodies. According to this approach, first, the abilityof any desired antibody to bind to essentially every possible sequenceof two amino acids that naturally appear in proteins is tested. Uponidentification of a pair of amino acids with detectable binding of theantibody, the sequence surrounding those two amino acids isprogressively and systematically varied, by the inclusion of each of thenaturally occurring amino acids as well as some amino acids not found innatural proteins, until continued testing of antibody binding identifiesa short peptide displaying an epitope with sufficient affinity for theselected antibody to be used for the desired purpose.

Thus, the approach of this invention of alloimmunization with epitopesof zona proteins is expected to have wide application in the design offuture contraceptive vaccines for the control of mammalian populations.

The present invention can be illustrated by the use of the followingnon-limiting examples.

EXAMPLE 1

Determination of the Primary Structure of Mouse and Human Zona PellucidaProteins by Cloning and Characterizing the Mouse and Human ZP3 and ZP2Genes.

A cDNA library was made from poly(A)⁺ RNA isolated from mouse ovariestissues using techniques standard to the field (Ringuette et al., Proc.Natl. Acad. Sci. USA 83:4341-45 (1986)). Eco RI linkers were added tothe ends of the cDNAs and the library was cloned into Eco RI site oflambda gt11. The library was packaged and used to infect E. coli Y1090cells which were mixed with agarose and plated in agar-filled petridishes using standard techniques. The lytic phase was induced by atemperature shift from 37° C. to 42° C. Nitrocellulose filters,impregnated with isopropyl β-D-thiogalactoside, were used to induceexpression of β-galactosidase fusion proteins by λgt11 recombinants.Those containing ZP2 or ZP3 epitopes were detected with a rabbitantisera that had been raised against heat solubilized mouse zonaepellucidae. The positive clones were plaque purified and tested fortheir ability to express fusion proteins that reacted with ratmonoclonal antibodies specific to either ZP2 or ZP3. Two λgt11recombinants reacted with a monoclonal antibody specific to ZP3(Ringuette et al., supra (1986); Ringuette et al., Dev. Biol. 127:287-95(1988)) and one λgt11 recombinant reacted with a monoclonal antibodyspecific to ZP2 (Liang et al., Mol. Cell. Biol. 10:1507-15 (1990)).

Mouse ZP3: The cDNA insert from a single λgt11 clone was subcloned(pZP3.1) and used to rescreen the library to obtain additional cDNAs.The 5' most 46 nt were determined from a genomic clone and thetranscription initiation site was determined by procedures standard tothe field (Ringuette et al., supra (1988)). These sequences were used todetermine the structure of the mouse ZP3 mRNA and the resultant protein.The ZP3 mRNA is a 1317 nt polyadenylated transcript that contains asingle open reading frame encoding a 424 amino acid polypeptide chainwith a predicted mass of 46,307 Da. The identity of the cDNA clone wasconfirmed by comparison of its deduced amino acid sequence with that ofa 20 amino acid sequence obtained from an internal peptide of purifiedZP3 protein. A predicted signal peptidase cut site after amino acid 17would result in a polypeptide with a mass of 43,943 Da (Ringuette etal., supra 1988). The 83,000 Da mass of the native, secreted ZP3sulfated glycoprotein reflects post-translational modifications of thepolypeptide chain. Additional characteristics of this protein have beennoted above.

Mouse ZP2: The cDNA insert from a single λgt11 clone was subcloned(pZP2.1) and used to rescreen the library to obtain additional cDNAsthat contained sequences that encoded the entire polypeptide chain(Liang et al., Mol. Cell. Biol. 10:1507-15 (1990)). The 5' most 21 ntwere determined from a genomic clone, and the transcription initiationsite was determined by procedures standard to the field. These sequenceswere used to determine the structure of the mouse ZP2 mRNA and theresultant protein. The ZP3 mRNA is a 2201 nt polyadenylated transcriptthat contains a single open reading frame encoding a 713 amino acidpolypeptide chain with a predicted mass of 80,217 Da. The identity ofthe clone was confirmed by comparison of its deduced amino acid sequencewith that of a 16 amino acid sequence from a N-terminal peptide and withthat of a 10 amino acid sequence obtained from an internal peptide ofpurified ZP2 protein. The first 34 amino acids represent a signalpeptide, the cleavage of which would result in a polypeptide with a massof 76,373 Da (Liang et al., supra (1990)). The 120-140,000 Da mass ofthe native, secreted ZP2 sulfated glycoprotein reflectspost-translational modifications of the polypeptide chain.

EXAMPLE 2

Conservation of the Zona Pellucida Genes Among Mammals, SpecificallyMouse and Human: Mouse Zp-3 genomic clones were isolated from a λJ1library containing mouse B10A genomic DNA inserts by screening withmouse ZP3 cDNA (Chamberlin et al., Dev. Biol. 131:207-14 (1989)).Characterization of two overlapping clones revealed that the single copyZp-3 gene contains 8 exons spanning 8.6-kbp. Exon sequences confirmedthe previously described coding region of the ZP3 mRNA (Ringuette etal., Dev. Biol. 127:287-95 (1988); Chamberlin et al., supra (1989)). Amouse Zp-2 genomic clone was isolated from the same λJ1 library byscreening with mouse ZP2 cDNA (Liang et al., Mol. Cell. Biol. 10:1507-15(1990)). The single copy Zp-2 gene contains 18 exons spanning 12.1-kbp.Exon sequences confirmed the previously described coding region of theZP2 mRNA (Liang et al., supra (1990)).

DNA was isolated from seven mammalian species: mouse, rat, rabbit, dog,pig, cow and human. After digestion with a restriction enzyme (e.g., BamH1) and transfer to a membrane by Southern blotting, the DNAs wereprobed with mouse ZP3 cDNA using standard techniques. Although strongerhybridization was detected with rat, dog, cow and human than with rabbitand pig DNA, cross-hybridization was detected with DNA from allmammalian species (Ringuette et al., Proc. Natl. Acad. Sci. USA83:4341-45 (1986)). Similar results were obtained using ZP2 cDNA probes.Mouse ZP3 cDNA probes cross-hybridized with rat and rabbit ovarianpoly(A)⁺ RNA on Northern blots and all three species have transcripts ofsimilar size (Ringuette et al., supra (1988)). Taken together, thesedata suggest that the zona genes are well conserved among mammals. Tofurther substantiate this hypothesis, human ZP2 and human ZP3 genes andtheir RNA transcripts were isolated and characterized.

Human ZP3: A Charon 4A human genomic library was screened with mouse ZP3cDNA under low stringency to allow cross-hybridization with theheterologous probe (Chamberlin et al., Proc. Natl. Acad. Sci. USA87:6014-18 (1990)). A single recombinant phage was isolated andcharacterized. This clone contained exons 1-5 of the human ZP3 gene. Theremaining 6-8 exons were cloned from genomic DNA using the polymerasechain reaction and oligonucleotide primers from human exons 6 and 8(determined from human ZP3 cDNA, see below). The human ZP3 genescontains 8 exons, the sizes of which have near identity with those ofmouse Zp-3, and the human gene spans approximately 18.3-kbp (Chamberlinet al., supra (1990)).

Poly (A)⁺ RNA was isolated from a human ovary and used in a RT-PCRreaction (reverse transcription to make a single strand cDNA template,followed by exon specific oligonucleotide primers in the polymerasechain reaction) to construct full-length cDNA clones representative ofthe human ZP3 transcript) (Chamberlin et al., supra (1990)). The humanZP3 transcript has a single 1272 nt open reading frame, the nucleic acidsequence of which is 74% identical to that of the mouse ZP3 transcript.The human transcript encodes a 424 amino acid polypeptide ZP3 proteinwith a calculated molecular mass of 47,032 Da that is 67% identical tothat of the mouse ZP3 protein. The hydropathicity profiles of the humanand mouse ZP3 proteins are remarkably similar and reflect the conservednature of the allowable amino acid substitutions (Chamberlin et al.,supra (1990)). Additional characteristics of this protein have beennoted above.

Human ZP2: A Charon 4A human genomic library was screened with mouse ZP2cDNA under low stringency to allow cross-hybridization with theheterologous probe (Liang et al., Dev. Biol. 156: 399-408 (1993)). Threeoverlapping recombinant phages were isolated and characterized. Theseclones contained the entire 14.0-kbp human ZP2 locus which is made up of19 exons. Overall, these coding regions are 70% identical to those ofmouse Zp-2. In addition, human ZP2 contains an extra exon of 84 bp (exon18) that is not found in mouse ZP2 cDNA. Sequence analysis of mouse Zp-2intron 17 revealed a region of 76 bp that shares a 70% sequence homologywith human ZP2 exon 18 (Liang et al., supra (1993)).

Poly (A)⁺ RNA was isolated from a human ovary and used in a RT-PCRreaction (reverse transcription to make a single strand cDNA template,followed by exon specific oligonucleotide primers in the polymerasechain reaction) to construct cDNA clones representative of the human ZP2transcript) (Liang et al., supra (1993)). In addition, human ovarianmRNA was used in the construction of an ovarian cDNA library using theUni-ZAP cDNA library construction system (stratagene). The library wasscreened with the aforementioned human ZP2 cDNA probes to isolateadditional cDNA clones that, together with those obtained with theRT-PCR, represented near full-length cDNAs. The nucleic acid sequence ofthese clones revealed that the human ZP2 transcript has a single 2235 ntopen reading frame that is 74% identical to that of the mouse ZP2transcript. The human transcript encodes a 745 amino acid ZP2 proteinwith a calculated molecular mass of 82,356 Da that is 60.7% identical tothat of the mouse ZP2 protein (Liang et al., supra (1993)). Thehydropathicity profiles of the human and mouse ZP2 proteins areremarkably similar and reflect the conserved nature of the allowableamino acid substitutions. Additional characteristics of this proteinhave been noted above.

These two examples demonstrate that the primary amino acid sequence ofthe zona pellucida proteins (ZP3, ZP2, ZP1) can be deduced from clonedzona genes (cDNAs and/or genomic clones). This data would not otherwisebe available because the paucity of biological material makes impossiblethe direct determination of the zona protein sequences. Furthermore,this example demonstrates that the conservation of the zona genes amongmammals permit the zona genes of one species (e.g. mouse) to be used toclone and characterize the zona genes of another species (e.g. human).Cross-hybridization data to genomic DNA from seven mammalian speciesfurther indicate that a similar strategy can be used to determine theprimary protein structures of the zona proteins from any mammal.

Further, this invention provides cDNA and genomic clones that can beused to express recombinant zona proteins of mouse and human ZP2 and ZP3in their entirety or in parts thereof. It will be obvious to those inthe field that this can be done using a variety of viral or plasmidbased vectors in a variety of prokaryotic and eukaryotic cell lines andin the production of transgenic animals. Such recombinant zona proteinsmay be of use as diagnostic reagents for the assessment of malefertility or lack thereof and for providing sufficient amounts of zonaproteins for further biochemical characterization of structure-functioncorrelates of the zona proteins.

EXAMPLE 3

Identification of ZP3 and ZP2 Peptides Capable of Eliciting Antibodiesthat Bind to the zona Pellucida Protein in the Same Species

Prior to this invention, it had not been demonstrated that a peptidecomprised of a portion of a zona protein from a particular species couldelicit antibodies in that same species that would bind to the nativezona pellucida structure and prevent fertilization. The success ofdemonstrating the efficacy of this approach is based on two aspects ofthis invention: the determination of the primary amino acid sequence ofthe mouse and human ZP2 and ZP3 proteins by cloning the cognate genes(Ringuette et al., Dev. Biol. 127:287-95 (1988); Chamberlin et al., Dev.Biol. 131:207-14 (1989); Liang et al., Mol. Cell. Biol. 10:1507-15(1990); Chamberlin et al., Proc. Natl. Acad. Sci. USA 87:6014-18 (1990);Liang et al., Dev. Biol. 156:399-408 (1993)), and the identification ofcandidate regions on the zona proteins to test the efficacy of thiscontraceptive strategy. In addition, the determination that the zonapellucida proteins are well conserved between mouse and human indicatesthat the three-dimensional structures of ZP2 and ZP3 in differentmammalian species will have near identity. Thus, regions of the zonaproteins identified as potential vaccine candidates in one species (e.g.mouse) will be effective in other species (e.g. humans). These regionsneed not have identical amino acid sequence but need only be located inthe homologous region of the zona pellucida matrix of each particularspecies.

As indicated above, once the primary amino acid sequence of a protein isknown, a variety of strategies can be used to identify candidatepeptides for testing as contraceptive vaccines. An example of onestrategy is provided in the invention.

The first candidate peptide was identified on mouse ZP3 by screening anepitope expression library derived from a ZP3 cDNA with a monoclonalantibody specific to the ZP3 protein.

A 1.0 kb cDNA known to contain the epitope recognized by the anti-ZP3monoclonal antibody (Ringuette et al., supra (1986)) was cut into randomfragments which were size selected (200 bp) and cloned into the λgt11expression vector. More specifically, the cDNA insert of pZP3.1 wasdigested with DNase in the presence of 15 mM MgCl₂ and 200 bp sizeselected fragments (V. Mehra, D. Sweetwer and R. A. Young, Proc. Natl.Acad. Sci. USA 83: 7013 (1986)) were ligated into Lambda ZAP(Strategene). E. coli BB4 cells were infected with the un-amplifiedepitope library and screened (Ringuette et al., supra (1986)), with ananti-ZP3 monoclonal antibody (East et al., Dev. Biol. 109: 268 (1985)).Positive clones were plaque purified and the sequence of the insert DNAwas determined from isolated plasmid DNA (F. Sanger, S. Nicklen, et al.,Proc. Natl. Acad. Sci. USA 74:5463 (1977)).

A synthetic peptide displaying an epitope for a contraceptive antibody.The nucleic acid sequence of the cDNA inserts from 8 positive clones wasdetermined (FIG. 7A). The 24 nucleotides common to the eight clones codefor a seven amino acid peptide which must contain the epitope recognizedby the antibody (FIG. 7B). The peptide represents amino acids 336-342which is immediately adjacent to the most hydrophilic portion of ZP3(FIG. 7C). A 16 amino acid peptide (ZP3 amino acids 328-343) containingthe epitope (NH₂-CYS-SER-ASN-SER-SER-SER-SER-GLN-PHE-GLN-ILE-HIS-GLY-PRO-ARG-GLN-COOH)was synthesized (Merrifield, R. B., J. Amer. Soc., 85: 2149 (1963)) on aModel 430A, Applied Biosystems Solid Phase Synthesizer, deprotected andreleased from the phenylacetamidomethyl resin with anhydrous hydrogenfluoride containing 10% anisole and 10% thiophenol at 0° C. for 2 hr.The crude peptide was purified by HPLC on a Vydac C4 column andconjugated to keyhole limpet hemocyanin by coupling the amino terminalcysteine to KLH through a maleimido linkage (Lerner, R. A. et al., Proc.Natl. Acad. Sci. USA, 78:3403 (1981)).

Immunogenicity of the synthetic peptide vaccine. Sixteen NIH random bredSwiss mice were immunized intraperitoneally with 100 μg of the ZP3peptide-KLH conjugate (1 mg/ml) in an equal volume of complete Freund'sadjuvant and then boosted at 10-14 day intervals with 100 μg ofconjugated peptide in incomplete Freund's adjuvant. Circulatinganti-zona pellucida antibodies were detected using solubilized wholezona in an ELISA. Flexible ELISA plates were coated with purified, acidsolubilized zona (J. D. Bleil and P. M. Wassarman, J. Cell Biol.102:1363 (1986)) at 100 ng per well, blocked with 1% bovine serumalbumin in Tris HCl, pH 7.5, 0.15M NaCl (TBS), and incubated with seradiluted 1:10⁴ in the same. The plates were washed several times withTBS/1% Tween-20, incubated with horse radish peroxidase (HRP) conjugatedgoat anti-mouse antibody, washed as before, and developed using aHorseradish Peroxidase Substrate Kit (Bio-Rad). The response wasquantified by measuring absorbance at 414nm.

A plateau level of the average response was reached after fiveimmunizations. It should be noted that there was variation of the amountof circulating anti-zona pellucida antibodies among the animals with thedifference between the high and low responders being almost six-fold.Control animals were immunized with KLH alone using an identical regimenand had no detectable circulating anti-zona antibodies.

The reactivity of sera from immunized animals with individual zonaproteins was analyzed using Western blots of purified zonae separated bySDS-PAGE. Isolated mouse zona were acid solubilized and separated bySDS-PAGE using 10% acrylamide (U. K. Laemmli, Nature 227:680 (1970)).Proteins were transferred to nitrocellulose (W. N. Burnette, Analyt.Biochem. 112:195 (1980)) and the filters soaked in TBS/1% BSA. Sera orantibodies were diluted in TBS/1% BSA/0.1% Tween and individual laneswere probed with: pre-immune sera diluted 1:50; immune sera from KLHimmunized mice diluted 1:50; immune sera from ZP3 peptide-KLH immunizedmice diluted 1:50; rat anti-mouse ZP3 monoclonal antibody (East et al.,Dev. Biol. 109:268-73 (1985)) diluted 1:50; and rabbit anti-mouse zonapellucida polyclonal antisera (East et al., supra (1985)) diluted 1:50.Filters were washed in TBS/0.1% Tween and incubated with HRP-labeledsecond antibody of the appropriate specificity (Jackson Immunoresearch)diluted 1:1000 in TBS/BSA/Tween. Nitrocellulose-bound antibodies werevisualized using 4-chloro-1-naphthol.

Sera from animals immunized with the ZP3 peptide-KLH conjugate reactedwith a single zona protein which co-migrated with ZP3. No reaction withany of the zona proteins was detected with pre-immune or control sera.

To determine whether anti-peptide antibodies recognize zona in itsnative state as well as in acid-solubilized and SDS-denaturedpreparations, sera from experimental and control animals were used tostain unfixed frozen sections of mouse ovary. Ovaries were removed andimmediately frozen in Tissue-Tek O.C.T. Compound (Lab-Tek Products) ondry ice. Five μm sections were mounted on gelatin coated slides, treatedwith 1% BSA in PBS for 15 min at 20° C. and rinsed in PBS. Sections weretreated for one hour with undiluted serum from immunized mice, rinsed inPBS and stained for 30 min at 20° C. with FITC-conjugated goatanti-mouse IgG (Jackson ImunoResearch Laboratories) diluted 1:50 inPBS/BSA. Sections were rinsed with PBS, mounted in Fluormount-S(FisherBiotech) and photographed using Ektachrome 200 film.

Using a fluorescein-conjugated second antibody, mouse antibodies fromexperimental mice were detected binding to the zonae surroundingdeveloping oocytes, indicating that the circulating anti-zona antibodiesare capable of binding native ZP3 protein. There was no detectablefluorescence of sections stained with sera from control mice.

As evidence that alloimmunization with a zona pellucida peptide is notlimited to the ZP3 protein, a second candidate peptide was identified onmouse ZP2 by screening an epitope expression library derived from a ZP2cDNA with a monoclonal antibody specific to the ZP2 protein using thetechniques described above. Specifically, a 0.9-kbp cDNA (pZP2.1) knownto contain the epitope recognized by an anti-ZP2 monoclonal antibody(Liang et al., Mol. Cell. Biol. 10:1507-15 (1990)) was digested withDNAse to create random fragments that were cloned into Lambda ZAP(Stratagene) to create an expression epitope library. The library wasscreened with the monoclonal antibody specific to mouse ZP2 and thenucleic acid sequence of positive clones was determined. The 54 bpcommon to the positive clones must encode the epitope recognized by theantibody. The peptide represents amino acids 114-129 which arecoincident with the major hydrophilic portion of ZP2 (FIG. 8).

The 17 amino acid peptide (ZP2 amino acids 114-129) containing theepitope (NH₂-Ile-Arg-Val-Gly-Asp-Thr-Thr-Thr-Asp-Val-Arg-Tyr-Lys-Asp-Asp-Met-COOH)was synthesized by Merrifield solid phase synthesis (see above) with anN-terminal cysteine with which it was coupled to keyhole limpethemocyanin. Female mice immunized intraperitoneally with 100 μg of theZP3 peptide-KLH conjugate (1 mg/ml) in equal volume of complete Freund'sadjuvant and then boosted at 10-14 day intervals with 100 μg ofconjugated peptide in incomplete Freund's adjuvant. Circulatinganti-zona antibodies were detected in an ELISA as described above. After6 immunizations, four of five female mice developed anti-zona antibodiesat titers comparable to those immunized with the ZP3 peptide. These datademonstrate the ability to the ZP2 peptide to elicit antibodies thatcross-react with zona pellucida from the same species

EXAMPLE 4

A Contraceptive Vaccine Comprising a Synthetic Peptide with a ZP3Epitope

To determine if the circulating anti-ZP3 antibodies were of sufficienttiter to bind to the zonae surrounding growing oocytes of theexperimental mice, plastic embedded sections of ovaries isolated fromfour females immunized with ZP3-KLH conjugate were stained with horseradish peroxidase (HRP) conjugated anti-mouse antibody. Dissectedovaries were fixed for one hour in 1% glutaraldehyde, rinsed in PBS andembedded in JB4 plastic. Endogenous antibody was detected in 4 μmsections using an anti-mouse streptavidin-HRP kit (Zymed).

Mouse anti-zona pellucida antibodies were observed coating the zonae ofthe oocytes in the sections examined. There were no detectable anti-zonaantibodies in ovaries isolated from four control (KLH alone injected)mice. The ovarian sections of both the treated and control animalscontained only normal follicles and cell types with no evidence ofinflammation or cellular cytotoxicity. The antisera of the ZP3-KLHimmunized animals did not react with other mouse tissue including brain,liver, spleen, kidney, heart, lung, intestine, testis or muscle (datanot shown) which indicates that immunization with the peptide conjugateelicits a response that is specific for the zona pellucida.

Effectiveness of the synthetic peptide vaccine for contraception. Thefertility of the remaining 12 experimental and 12 control mice wastested by mating them continuously with proven males. Two weeks afterthe last immunization, proven males were individually and continuouslycaged with experimental and control mice at a ratio of 1:1. Thepercentage of animals having given birth to a litter versus the durationof continuous mating was compared for animals injected with ZP3peptide-KLH and KLH alone. The titer of anti-ZP antibodies of threegroups of ZP3 peptide-KLH immunized mice at the beginning of the matingperiod were averaged and, in order of increasing average titers, were asfollows: group 1, gave birth within 1 month (3 animals); group 2, gavebirth between 4 and 7 months (3 animals); and group 3, did not givebirth to litters within the 9 month study (6 animals).

In summary, all of the control (KLH alone injected) mice gave birth tolitters within three and a half weeks of the introduction of males.Three of the experimental, ZP3 peptide-KLH injected mice also gave birthwithin this period. These mice were among those that had the lowesttiters (<0.2 A₄₁₄ units) of anti-zona antibodies prior to mating. In theremainder of the experimental mice, a contraceptive effect was observedthat lasted between 16 and 36 weeks at which time the study wasterminated. Three of these animals gave birth to litters after 16 to 24weeks and had intermediate anti-zona antibody titers. The remaininganimals which remained infertile for the duration of the study had thehighest initial titers and even 9 months after the last immunization haddetectable circulating anti-zona antibodies.

The litter sizes of the ZP3-KLH treated animals which eventually becamefertile ranged from 1-5 pups (average 2.8) whereas those treated withKLH alone had litters of 1-9 pups (average 5.2). Both groups had fewerthan the normal 7-14 pups (average 10) which may be due, in part, to theadverse effects of intra-peritoneal administration of Freund's adjuvanton fecundity. In addition, the smaller litters of the KLH-ZP3 treatedanimals could be accounted for by the observed persistent low levels ofcirculating anti-zona antibodies some of which were detected binding tothe zonae surrounding their intra-ovarian oocytes. Despite the presenceof these low levels of anti-zona antibodies, these animals, whenre-mated, gave birth to litters within three and a half weeks.

The foregoing invention has been described in some detail for purposesof clarity and understanding. It will also be obvious that variouscombinations in form and detail can be made without departing from thescope of the invention.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES:  12    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  2266              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  doub - #le              (D) TOPOLOGY:  unknown    -     (ii) MOLECULE TYPE: cDNA    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: human              (B) STRAIN:              (C) INDIVIDUAL ISOLATE:              (D) DEVELOPMENTAL STAGE:              (E) HAPLOTYPE:              (F) TISSUE TYPE:              (G) CELL TYPE:              (H) CELL LINE:              (I) ORGANELLE:    -     (ix) FEATURE:              (A) NAME/KEY: ZP2              (B) LOCATION:              (C) IDENTIFICATION METHOD:    #human ZP2 cDNATHER INFORMATION:    -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:1:    #    40            GAGG AGGCTCTTGG AGTCCCTCAG    #    80            CTGG AGCACCTACA GGTCGATTTC    #   120            GTGA CTTCAGGGAA CTCCATAGAT    #   160            ATCC TGCCTTTCCA GGCACTGTCA    #   200            AATA ACAGTGGAGT TCCCAAGCAG    #   240            TGGC ATGCATCTGT GGTGGATCCT    #   280            CGAA CTGCACTTAC ATCCTGGACC    #   320            GAGG GCTACCTATG ATAACTGTAC    #   360            GGAC ACCAGATGAC CATCAGAGTC    #   400            CCTT AAGACACGGA GCTGTCATGT    #   440            AGCT ATGCAAGTAG AAGAGACCCA    #   480            ACAA TCTGCCAGAA GGATTTCATG    #   520            GGGT CTTCTCTGGC TTGGCTGACG    #   560            AGTT CAGATGGGAT GGAGCATTGA    #   600            AGAG CCAAAACTCT GACCCTGCCA    #   640            GCTT CAGCCTCTTG ATTGACAACC    #   680            TGTG CCATTCAATG CCACTGGAGT    #   720            GGTA ACAGTCATCT CTACATGGTG    #   760            TTAT ATCTCCTGGA CAGAAGGTGA    #   800            TATT TGTGCACCAG ATCCTGTGAC    #   840            ATGA CTCTCACCAT ACCAGAGTTT    #   880            CTGT GAGCTTTGAA AACCAGAACA    #   920            GCAT GACAATGGAA TTGATCTAGA    #   960            AAAT TGCATTTCAG CAAAACTCTG    #  1000            CTGA AAAATGCCTA CTCCATCAGT    #  1040            CAAG CTGACCTTTC TCCTTCGGCC    #  1080            GTGA TCTATCCTGA GTGTCTCTGT    #  1120            TAGT TACAGGGGAG CTGTGCACCC    #  1160            CGTC GAGGTCTACA GCTACCAAAC    #  1200            CTGG GTACTCTGAG GGTGGGAAAC    #  1240            TCTT TGAGGCTCAG TCTCAGGGGC    #  1280            ACCC CTGAATGGAT GTGGAACGAG    #  1320            GATA AAGTCGTCTA TGAAAACGAA    #  1360            CGGA TTTTCCTCCA AGCAAAATAT    #  1400            CAGA ATGACAGTGA AGTGTTCTTA    #  1440            CTAC TAAACATCAA CGTTGAAAGC    #  1480            CCTC AGTGAAGTTG GGTCCATTTA    #  1520            CTAC CCAGATAATT CCTACCAACA    #  1560            GAGT ACCCTCTAGT GAGATTCCTC    #  1600            TGGA AGTGAGAGTC CTAAACAGGG    #  1640            GCTG GTCTTAGATG ACTGCTGGGC    #  1680            CCAG ACTCTTTCCC CCAGTGGAAC    #  1720            GTGC ATATGACCTG GACAACTACC    #  1760            AGTC GGCTCCTCTG TGACCCATCC    #  1800            TTTG ACATGAAGGC TTTTGCCTTT    #  1840            TGCT CTCTAGCCTG GTCTACTTCC    #  1880            CTGT AATCGACTCT CCCCTGACTC    #  1920            ACCT GCCCTGTGTC CTCTAGGCAC    #  1960            CCAC TGAAGCAGAG AAAATGACAG    #  2000            CATT CTCCTGTTGT CAGATGACTC    #  2040            GGCT CATCTGATCT AAAAGCAAGT    #  2080            AGAG TAGGAGTGAA ACAGGGGAGG    #  2120            TGCT ATGGACACCA AAGGGCACAA    #  2160            GGTT CCAAAGCTGT GGCTGCTGTG    #  2200            TGGT GGCAACTCTA GGCTTCATCT    #  2240            AAGG ACTGTGTCAA ATCACTAAAT    #            2266  CAGT CAAAAT    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  2201              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  doub - #le              (D) TOPOLOGY:  unknown    -     (ii) MOLECULE TYPE: cDNA    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: mouse              (B) STRAIN:              (C) INDIVIDUAL ISOLATE:              (D) DEVELOPMENTAL STAGE:              (E) HAPLOTYPE:              (F) TISSUE TYPE:              (G) CELL TYPE:              (H) CELL LINE:              (I) ORGANELLE:    -     (ix) FEATURE:              (A) NAME/KEY: ZP2              (B) LOCATION:              (C) IDENTIFICATION METHOD:    #mouse ZP2 cDNATHER INFORMATION:    -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:2:    #    40            TGGT ACCTTCCAAC ATGGCGAGGT    #    80            TGTA AGCTCTCCGT GCGGCAGGAG    #   120            TCCC TCTTATTCAC CCTTGTGACT    #   160            GCCT TCCTCAGTCC GAGAATCCTG    #   200            CATT TGTGACAAAG ACGAAGTGAG    #   240            AGAT TTGACATGGA AAAATGGAAT    #   280            CCCT TGGTAGTGAA ATTTTGAACT    #   320            CTTG GAAAGGTTCG TCCTGAAGTT    #   360            ACTA TAAAAGTGGT TGGTGGATAC    #   400            TGGG GGACACCACC ACTGATGTGA    #   440            GTAT CATTTCTTCT GTCCAGCTAT    #   480            GAGA TTTCAGAAAT TGTTGTCTGC    #   520            CTTT TTCTTTCCCA CAACTTTTCT    #   560            AAAC CAGAATGTAT CTGAGATGGG    #   600            GGCA ATGGTACAAG AGCCCACATT    #   640            CCAT AGTACAAGGA TTTAATCTTC    #   680            AGTG ACTCTCCACG TGCCAGCCAA    #   720            CACT ATGTGCAAGA GAGCAGCTAT    #   760            TGGA GCTCTTGTTC TCAACCACTG    #   800            CTCA TCACACGCTA TCTGCGCACC    #   840            TGTA ATGCTACACA CATGACTCTC    #   880            CTGG GAAGCTAGAG TCTGTGGACT    #   920            CCCT GAGGACCAAT GGCATGCCAA    #   960            GCAA CAAATGGCTT GAGATTGAAT    #  1000            TGAA AACTAAACCC TCTGAAAAAT    #  1040            CTAC CTCTCTTCAC TCAAGCTGAC    #  1080            AACA TGCTATCCAC AGTGATAGAT    #  1120            AGTC ACCAGTCTCT ATAGATGAAC    #  1160            GTTT ATGGACTTTG AGGTCTACAG    #  1200            GCAC TGAACCTGGA CACCCTCCTG    #  1240            GCCA GCCTATTTTC AAGGTGCAGT    #  1280            GTTT CACATACCTC TGAATGGATG    #  1320            TTTG AAGGTGATAA AGTCATCTAT    #  1360            CTCT CTGGGAAAAC CCACCCTCCA    #  1400            CAGC GAGTTCAGGA TGACAGTAAG    #  1440            GACA GTATGCTACT AAATGCCCAT    #  1480            CTCC AGAGGCCTTT GTAAAGCCAG    #  1520            CCTA CAAACATACC CAGACCAATC    #  1560            AGGA AGGATGAGTA CCCTCTAGTG    #  1600            CAAT CTACATGGAA GTGAAGGTCT    #  1640            CAAC ATCAAGCTGG TCTTAGATGA    #  1680            TCTG AGGACCCGGC CTCTGCGCCT    #  1720            TGGA TGGCTGTGAA TATGAACTGG    #  1760            TTTC CACCCAGCTG GCTCCTCTGC    #  1800            TACC AGAGGTTTGA TGTGAAGACT    #  1840            AGGC ACGGGGGCTC TCCAGCCTGA    #  1880            TGCC TTGATCTGTA ACCAAGTCTC    #  1920            TGCT CTGTGACTTG CCCTGCATCA    #  1960            AGGC CAACAAAGAA GACACAATGA    #  2000            ACCT ATTCTCTTGC TGTCAGATGT    #  2040            GTTG ACCCCAGCAG CTCTGAGATT    #  2080            CCAA GGATATTGCT TCTAAAACAC    #  2120            ACTA GTGGGCTCAG CTGTCATTCT    #  2160            CTGT ATAAGAAAAG AACTATAAGG    #  2200            ACTT GCAAATAAAG AGACTGCAGT    #             2201    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  1299              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  doub - #le              (D) TOPOLOGY:  unknown    -     (ii) MOLECULE TYPE: cDNA    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: human              (B) STRAIN:              (C) INDIVIDUAL ISOLATE:              (D) DEVELOPMENTAL STAGE:              (E) HAPLOTYPE:              (F) TISSUE TYPE:              (G) CELL TYPE:              (H) CELL LINE:              (I) ORGANELLE:    -     (ix) FEATURE:              (A) NAME/KEY: ZP3              (B) LOCATION:              (C) IDENTIFICATION METHOD:    #human ZP3 cDNATHER INFORMATION:    -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:3:    #    40            CTGA GCTATAGGCT CTTCATCTGC    #    80            GTAC TGAGCTGTGC TACCCCCAAC    #   120            GGGT GGAGCCAGCC ATCCTGAGAC    #   160            CTGG TGGAGTGTCA GGAGGCCACT    #   200            GCAA AGACCTTTTT GGCACCGGGA    #   240            TGAC CTCACCTTGG GCCCAGAGGC    #   280            TCCA TGGACACAGA AGATGTGGTC    #   320            TCCA CGAGTGTGGC AACAGCATGC    #   360            CCTG GTGTACAGCA CCTTCCTGCT    #   400            GTGG GAAACCTGTC CATCGTGAGG    #   440            TTCC CATCGAGTGC CGCTACCCCA    #   480            CAGC CAGGCCATCC TGCCCACCTG    #   520            ACGG TGTTCTCAGA GGAGAAGCTG    #   560            TGAT GGAGGAGAAC TGGAACGCTG    #   600            CTTC CACCTGGGAG ATGCAGCCCA    #   640            CACA CTGGCAGCCA CGTGCCACTG    #   680            ACTG CGTGGCCACA CCGACACCAG    #   720            TTAT CACACCATCG TGGACTTCCA    #   760            GGTC TCACTGATGC CTCTTCTGCA    #   800            CCGG GCCAGATACA CTCCAGTTCA    #   840            CTTT GCTAATGACT CCAGAAACAT    #   880            CACC TGAAGGTCAC CCTAGCTGAG    #   920            TCAA CAAGGCCTGT TCCTTCAGCA    #   960            GTTC CCAGTGGAAG GCCCGGCTGA    #  1000            AACA AAGGTGACTG TGGCACTCCA    #  1040            AGCC TCATGTCATG AGCCAGTGGT    #  1080            TAAC CGCAGGCATG TGACAGAAGA    #  1120            GGGC CACTGATCTT CCTGGACAGG    #  1160            TAGA GCAGTGGGCT TTGCCTTCTG    #  1200            GCTG GGCGTAGGCC TGGCTGTGGT    #  1240            ACTG CTGTTATCCT GGTTCTCACC    #  1280            CCTC CCACCCTGTG TCTGCTTCCG    #                 129 - #9    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  1317              (B) TYPE:  nucleic a - #cid              (C) STRANDEDNESS:  doub - #le              (D) TOPOLOGY:  unknown    -     (ii) MOLECULE TYPE: cDNA    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: mouse              (B) STRAIN:              (C) INDIVIDUAL ISOLATE:              (D) DEVELOPMENTAL STAGE:              (E) HAPLOTYPE:              (F) TISSUE TYPE:              (G) CELL TYPE:              (H) CELL LINE:              (I) ORGANELLE:    -     (ix) FEATURE:              (A) NAME/KEY: ZP3              (B) LOCATION:              (C) IDENTIFICATION METHOD:    #mouse ZP3 cDNATHER INFORMATION:    -     (xi) SEQUENCE DESCRIPTION:SEQ  ID NO:4:    #    40            TCCA GGCGGGACCA TGGCGTCAAG    #    80            TGTC TCCTGCTGTG TGGAGGCCCC    #   120            AGAC TCTGTGGCTT TTGCCGGGTG    #   160            GGGG TCCTCATCAC CTGTGAAGGT    #   200            GAAC TAGTGGTGAC TGTCAGTAGA    #   240            GGAA GCTGGTGCAG CCCGGGGACC    #   280            GGGT TGTCAGCCCC GGGTGTCCGT    #   320            AGGT TCAACGCCCA GTTGCACGAG    #   360            AGAT GACGAAAGAT GCCCTGGTGT    #   400            CCAC GACCCTCGCC CTGTGAGTGG    #   440            ACTA ACCGTGTGGA GGTACCCATT    #   480            GGCA GGGCAATGTG AGCAGCCACC    #   520            GGTT CCCTTCAGAG CCACTGTGTC    #   560            GCTT TCTCTCTTCG CCTGATGGAG    #   600            AGAA ATCGGCTCCC ACCTTCCACC    #   640            CCTC CAGGCAGAAG TCCAGACTGG    #   680            CAGC TGTTTGTGGA CCACTGCGTG    #   720            TGCC AGACCCGAAC TCCTCCCCCT    #   760            CTTC CACGGTTGCC TTGTGGATGG    #   800            TCGG CATTTCAAGT CCCCAGACCC    #   840            AGTT CACGGTGGAT GTATTCCATT    #   880            AAAT ACGCTCTACA TCACCTGCCA    #   920            GCTA ACCAGATCCC CGATAAGCTC    #   960            TCAA CAAGACTTCC CAGAGTTGGT    #  1000            TGCT GACATCTGTG ATTGCTGCAG    #  1040            AATT CAAGCTCTTC ACAGTTCCAG    #  1080            AGTG GTCCAAGCTA GTTTCTCGAA    #  1120            CGAT GAAGCTGATG TCACTGTAGG    #  1160            GGAA AGGCCAACGA CCAGACTGTG    #  1200            CTGC TCAAACCCCT GTGGCTCTTG    #  1240            AGTG GCATTCCTGA CCCTGGCAGC    #  1280            ACCA GGAAGTGTCA CTCCTCTTCC    #    1317          CGCA ATAAAAGAAG AAACTCA    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  745              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  unknown    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: human              (B) STRAIN:              (C) INDIVIDUAL ISOLATE:              (D) DEVELOPMENTAL STAGE:              (E) HAPLOTYPE:              (F) TISSUE TYPE:              (G) CELL TYPE:              (H) CELL LINE:              (I) ORGANELLE:    -     (ix) FEATURE:              (A) NAME/KEY: ZP2              (B) LOCATION:              (C) IDENTIFICATION METHOD:    #human ZP2 proteinR INFORMATION:    -     (xi) SEQUENCE DESCRIPTION:SEQ  ID NO:5:    - Met Ala Cys Arg Gln Arg Gly Gly Ser Trp Se - #r Pro    #                10    - Ser Gly Trp Phe Asn Ala Gly Trp Ser Thr Ty - #r Arg    #        20    - Ser Ile Ser Leu Phe Phe Ala Leu Val Thr Se - #r Gly    #35    - Asn Ser Ile Asp Val Ser Gln Leu Val Asn Pr - #o Ala    #            45    - Phe Pro Gly Thr Val Thr Cys Asp Glu Arg Gl - #u Ile    #    60    - Thr Val Glu Phe Pro Ser Ser Pro Gly Thr Ly - #s Lys    #                70    - Trp His Ala Ser Val Val Asp Pro Leu Gly Le - #u Asp    #        80    - Met Pro Asn Cys Thr Tyr Ile Leu Asp Pro Gl - #u Lys    #95    - Leu Thr Leu Arg Ala Thr Tyr Asp Asn Cys Th - #r Arg    #           105    - Arg Val His Gly Gly His Gln Met Thr Ile Ar - #g Val    #   120    - Met Asn Asn Ser Ala Ala Leu Arg His Gly Al - #a Val    #               130    - Met Tyr Gln Phe Phe Cys Pro Ala Met Gln Va - #l Glu    #       140    - Glu Thr Gln Gly Leu Ser Ala Ser Thr Ile Cy - #s Gln    145                 1 - #50                 1 - #55    - Lys Asp Phe Met Ser Phe Ser Leu Pro Arg Va - #l Phe    #           165    - Ser Gly Leu Ala Asp Asp Ser Lys Gly Thr Ly - #s Val    #   180    - Gln Met Gly Trp Ser Ile Glu Val Gly Asp Gl - #y Ala    #               190    - Arg Ala Lys Thr Leu Thr Leu Pro Glu Ala Me - #t Lys    #       200    - Glu Gly Phe Ser Leu Leu Ile Asp Asn His Ar - #g Met    205                 2 - #10                 2 - #15    - Thr Phe His Val Pro Phe Asn Ala Thr Gly Va - #l Thr    #           225    - His Tyr Val Gln Gly Asn Ser His Leu Tyr Me - #t Val    #   240    - Ser Leu Lys Leu Thr Phe Ile Ser Pro Gly Gl - #n Lys    #               250    - Val Ile Phe Ser Ser Gln Ala Ile Cys Ala Pr - #o Asp    #       260    - Pro Val Thr Cys Asn Ala Thr His Met Thr Le - #u Thr    265                 2 - #70                 2 - #75    - Ile Pro Glu Phe Pro Gly Lys Leu Lys Ser Va - #l Ser    #           285    - Phe Glu Asn Gln Asn Ile Asp Val Ser Gln Le - #u His    #   300    - Asp Asn Gly Ile Asp Leu Glu Ala Thr Asn Gl - #y Met    #               310    - Lys Leu His Phe Ser Lys Thr Leu Leu Lys Th - #r Lys    #       320    - Leu Ser Glu Lys Cys Leu Leu His Gln Phe Ty - #r Leu    325                 3 - #30                 3 - #35    - Ala Ser Leu Lys Leu Thr Phe Leu Leu Arg Pr - #o Glu    #           345    - Thr Val Ser Met Val Ile Tyr Pro Glu Cys Le - #u Cys    #   360    - Glu Ser Pro Val Ser Ile Val Thr Gly Glu Le - #u Cys    #               370    - Thr Gln Asp Gly Phe Met Asp Val Glu Val Ty - #r Ser    #       380    - Tyr Gln Thr Gln Pro Ala Leu Asp Leu Gly Th - #r Leu    385                 3 - #90                 3 - #95    - Arg Val Gly Asn Ser Ser Cys Gln Pro Val Ph - #e Glu    #           405    - Ala Gln Ser Gln Gly Leu Val Arg Phe His Il - #e Pro    #   420    - Leu Asn Gly Cys Gly Thr Arg Tyr Lys Phe Gl - #u Asp    #               430    - Asp Lys Val Val Tyr Glu Asn Glu Ile His Al - #a Leu    #       440    - Trp Thr Asp Phe Pro Pro Ser Lys Ile Ser Ar - #g Asp    445                 4 - #50                 4 - #55    - Ser Glu Phe Arg Met Thr Val Lys Cys Ser Ty - #r Ser    #           465    - Arg Asn Asp Met Leu Leu Asn Ile Asn Val Gl - #u Ser    #   480    - Leu Thr Pro Pro Val Ala Ser Val Lys Leu Gl - #y Pro    #               490    - Phe Thr Leu Ile Leu Gln Ser Tyr Pro Asp As - #n Ser    #       500    - Tyr Gln Gln Pro Tyr Gly Glu Asn Glu Tyr Pr - #o Leu    505                 5 - #10                 5 - #15    - Val Arg Phe Leu Arg Gln Pro Ile Tyr Met Gl - #u Val    #           525    - Arg Val Leu Asn Arg Asp Asp Pro Asn Ile Ly - #s Leu    #   540    - Val Leu Asp Asp Cys Trp Ala Thr Ser Thr Me - #t Asp    #               550    - Pro Asp Ser Phe Pro Gln Trp Asn Val Val Va - #l Asp    #       560    - Gly Cys Ala Tyr Asp Leu Asp Asn Tyr Gln Th - #r Thr    565                 5 - #70                 5 - #75    - Phe His Pro Val Gly Ser Ser Val Thr His Pr - #o Asp    #           585    - His Tyr Gln Arg Phe Asp Met Lys Ala Phe Al - #a Phe    #   600    - Val Ser Glu Ala His Val Leu Ser Ser Leu Va - #l Tyr    #               610    - Phe His Cys Ser Ala Leu Ile Cys Asn Arg Le - #u Ser    #       620    - Pro Asp Ser Pro Leu Cys Ser Val Thr Cys Pr - #o Val    625                 6 - #30                 6 - #35    - Ser Ser Arg His Arg Arg Ala Thr Gly Ala Th - #r Glu    #           645    - Ala Glu Lys Met Thr Val Ser Leu Pro Gly Pr - #o Ile    #   660    - Leu Leu Leu Ser Asp Asp Ser Ser Phe Arg Gl - #y Val    #               670    - Gly Ser Ser Asp Leu Lys Ala Ser Gly Ser Se - #r Gly    #       680    - Glu Lys Ser Arg Ser Glu Thr Gly Glu Glu Va - #l Gly    685                 6 - #90                 6 - #95    - Ser Arg Gly Ala Met Asp Thr Lys Gly His Ly - #s Thr    #           705    - Ala Gly Asp Val Gly Ser Lys Ala Val Ala Al - #a Val    #   720    - Ala Ala Phe Ala Gly Val Val Ala Thr Leu Gl - #y Phe    #               730    - Ile Tyr Tyr Leu Tyr Glu Lys Arg Thr Val Se - #r Asn    #   740    - His    745    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  713              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  unknown    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: mouse              (B) STRAIN:              (C) INDIVIDUAL ISOLATE:              (D) DEVELOPMENTAL STAGE:              (E) HAPLOTYPE:              (F) TISSUE TYPE:              (G) CELL TYPE:              (H) CELL LINE:              (I) ORGANELLE:    -     (ix) FEATURE:              (A) NAME/KEY: ZP2              (B) LOCATION:              (C) IDENTIFICATION METHOD:    #mouse ZP2 proteinR INFORMATION:    -     (xi) SEQUENCE DESCRIPTION:SEQ  ID NO:6:    - Met Ala Arg Trp Gln Arg Lys Ala Ser Val Se - #r Ser    #                10    - Pro Cys Gly Arg Ser Ile Tyr Arg Phe Leu Se - #r Leu    #        20    - Leu Phe Thr Leu Val Thr Ser Val Asn Ser Va - #l Ser    #35    - Leu Pro Gln Ser Glu Asn Pro Ala Phe Pro Gl - #y Thr    #            45    - Leu Ile Cys Asp Lys Asp Glu Val Arg Ile Gl - #u Phe    #    60    - Ser Ser Arg Phe Asp Met Glu Lys Trp Asn Pr - #o Ser    #                70    - Val Val Asp Thr Leu Gly Ser Glu Ile Leu As - #n Cys    #        80    - Thr Tyr Ala Leu Asp Leu Glu Arg Phe Val Le - #u Lys    #95    - Phe Pro Tyr Glu Thr Cys Thr Ile Lys Val Va - #l Gly    #           105    - Gly Tyr Gln Val Asn Ile Arg Val Gly Asp Th - #r Thr    #   120    - Thr Asp Val Arg Tyr Lys Asp Asp Met Tyr Hi - #s Phe    #               130    - Phe Cys Pro Ala Ile Gln Ala Glu Thr His Gl - #u Ile    #       140    - Ser Glu Ile Val Val Cys Arg Arg Asp Leu Il - #e Ser    145                 1 - #50                 1 - #55    - Phe Ser Phe Pro Gln Leu Phe Ser Arg Leu Al - #a Asp    #           165    - Glu Asn Gln Asn Val Ser Glu Met Gly Trp Il - #e Val    #   180    - Lys Ile Gly Asn Gly Thr Arg Ala His Ile Le - #u Pro    #               190    - Leu Lys Asp Ala Ile Val Gln Gly Phe Asn Le - #u Leu    #       200    - Ile Asp Ser Gln Lys Val Thr Leu His Val Pr - #o Ala    205                 2 - #10                 2 - #15    - Asn Ala Thr Gly Ile Val His Tyr Val Gln Gl - #u Ser    #           225    - Ser Tyr Leu Tyr Thr Val Gln Leu Glu Leu Le - #u Phe    #   240    - Ser Thr Thr Gly Gln Lys Ile Val Phe Ser Se - #r His    #               250    - Ala Ile Cys Ala Pro Asp Leu Ser Val Ala Cy - #s Asn    #       260    - Ala Thr His Met Thr Leu Thr Ile Pro Glu Ph - #e Pro    265                 2 - #70                 2 - #75    - Gly Lys Leu Glu Ser Val Asp Phe Gly Gln Tr - #p Ser    #           285    - Ile Pro Glu Asp Gln Trp His Ala Asn Gly Il - #e Asp    #   300    - Lys Glu Ala Thr Asn Gly Leu Arg Leu Asn Ph - #e Arg    #               310    - Lys Ser Leu Leu Lys Thr Lys Pro Ser Glu Ly - #s Cys    #       320    - Pro Phe Tyr Gln Phe Tyr Leu Ser Ser Leu Ly - #s Leu    325                 3 - #30                 3 - #35    - Thr Phe Tyr Phe Gln Gly Asn Met Leu Ser Th - #r Val    #           345    - Ile Asp Pro Glu Cys His Cys Glu Ser Pro Va - #l Ser    #   360    - Ile Asp Glu Leu Cys Ala Gln Asp Gly Phe Me - #t Asp    #               370    - Phe Glu Val Tyr Ser His Gln Thr Lys Pro Al - #a Leu    #       380    - Asn Leu Asp Thr Leu Leu Val Gly Asn Ser Se - #r Cys    385                 3 - #90                 3 - #95    - Gln Pro Ile Phe Lys Val Gln Ser Val Gly Le - #u Ala    #           405    - Arg Phe His Ile Pro Leu Asn Gly Cys Gly Th - #r Arg    #   420    - Gln Lys Phe Glu Gly Asp Lys Val Ile Tyr Gl - #u Asn    #               430    - Glu Ile His Ala Leu Trp Glu Asn Pro Pro Se - #r Asn    #       440    - Ile Val Phe Arg Asn Ser Glu Phe Arg Met Th - #r Val    445                 4 - #50                 4 - #55    - Arg Cys Tyr Tyr Ile Arg Asp Ser Met Leu Le - #u Asn    #           465    - Ala His Val Lys Gly His Pro Ser Pro Glu Al - #a Phe    #   480    - Val Lys Pro Gly Pro Leu Val Leu Val Leu Gl - #n Thr    #               490    - Tyr Pro Asp Gln Ser Tyr Gln Arg Pro Tyr Ar - #g Lys    #       500    - Asp Glu Tyr Pro Leu Val Arg Tyr Leu Arg Gl - #n Pro    505                 5 - #10                 5 - #15    - Ile Tyr Met Glu Val Lys Val Leu Ser Arg As - #n Asp    #           525    - Pro Asn Ile Lys Leu Val Leu Asp Asp Cys Tr - #p Ala    #   540    - Thr Ser Ser Glu Asp Pro Ala Ser Ala Pro Gl - #n Trp    #               550    - Gln Ile Val Met Asp Gly Cys Glu Tyr Glu Le - #u Asp    #       560    - Asn Tyr Arg Thr Thr Phe His Pro Ala Gly Se - #r Ser    565                 5 - #70                 5 - #75    - Ala Ala His Ser Gly His Tyr Gln Arg Phe As - #p Val    #           585    - Lys Thr Phe Ala Phe Val Ser Glu Ala Arg Gl - #y Leu    #   600    - Ser Ser Leu Ile Tyr Phe His Cys Ser Ala Le - #u Ile    #               610    - Cys Asn Gln Val Ser Leu Asp Ser Pro Leu Cy - #s Ser    #       620    - Val Thr Cys Pro Ala Ser Leu Arg Ser Lys Ar - #g Glu    625                 6 - #30                 6 - #35    - Ala Asn Lys Glu Asp Thr Met Thr Val Ser Le - #u Pro    #           645    - Gly Pro Ile Leu Leu Leu Ser Asp Val Ser Se - #r Ser    #   660    - Lys Gly Val Asp Pro Ser Ser Ser Glu Ile Th - #r Lys    #               670    - Asp Ile Ile Ala Lys Asp Ile Ala Ser Lys Th - #r Leu    #       680    - Gly Ala Val Ala Ala Leu Val Gly Ser Ala Va - #l Ile    685                 6 - #90                 6 - #95    - Leu Gly Phe Ile Cys Tyr Leu Tyr Lys Lys Ar - #g Thr    #           705    - Ile Arg Phe Asn His        710    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  424              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  unknown    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: human              (B) STRAIN:              (C) INDIVIDUAL ISOLATE:              (D) DEVELOPMENTAL STAGE:              (E) HAPLOTYPE:              (F) TISSUE TYPE:              (G) CELL TYPE:              (H) CELL LINE:              (I) ORGANELLE:    -     (ix) FEATURE:              (A) NAME/KEY: ZP3              (B) LOCATION:              (C) IDENTIFICATION METHOD:    #human ZP3 proteinR INFORMATION:    -     (xi) SEQUENCE DESCRIPTION:SEQ  ID NO:7:    - Met Glu Leu Ser Tyr Arg Leu Phe Ile Cys Le - #u Leu    #                10    - Leu Trp Gly Ser Thr Glu Leu Cys Tyr Pro Gl - #n Pro    #        20    - Leu Trp Leu Leu Gln Gly Gly Ala Ser His Pr - #o Glu    #35    - Thr Ser Val Gln Pro Val Leu Val Glu Cys Gl - #n Glu    #            45    - Ala Thr Leu Met Val Met Val Ser Lys Asp Le - #u Phe    #    60    - Gly Thr Gly Lys Leu Ile Arg Ala Ala Asp Le - #u Thr    #                70    - Leu Gly Pro Glu Ala Cys Glu Pro Leu Val Se - #r Met    #        80    - Asp Thr Glu Asp Val Val Arg Phe Glu Val Gl - #y Leu    #95    - His Glu Cys Gly Asn Ser Met Gln Val Thr As - #p Asp    #           105    - Ala Leu Val Tyr Ser Thr Phe Leu Leu His As - #p Pro    #   120    - Arg Pro Val Gly Asn Leu Ser Ile Val Arg Th - #r Asn    #               130    - Arg Ala Glu Ile Pro Ile Glu Cys Arg Tyr Pr - #o Arg    #       140    - Gln Gly Asn Val Ser Ser Gln Ala Ile Leu Pr - #o Thr    145                 1 - #50                 1 - #55    - Trp Leu Pro Phe Arg Thr Thr Val Phe Ser Gl - #u Glu    #           165    - Lys Leu Thr Phe Ser Leu Arg Leu Met Glu Gl - #u Asn    #   180    - Trp Asn Ala Glu Lys Arg Ser Pro Thr Phe Hi - #s Leu    #               190    - Gly Asp Ala Ala His Leu Gln Ala Glu Ile Hi - #s Thr    #       200    - Gly Ser His Val Pro Leu Arg Leu Phe Val As - #p His    205                 2 - #10                 2 - #15    - Cys Val Ala Thr Pro Thr Pro Asp Gln Asn Al - #a Ser    #           225    - Pro Tyr His Thr Ile Val Asp Phe His Gly Cy - #s Leu    #   240    - Val Asp Gly Leu Thr Asp Ala Ser Ser Ala Ph - #e Lys    #               250    - Val Pro Arg Pro Gly Pro Asp Thr Leu Gln Ph - #e Thr    #       260    - Val Asp Val Phe His Phe Ala Asn Asp Ser Ar - #g Asn    265                 2 - #70                 2 - #75    - Met Ile Tyr Ile Thr Cys His Leu Lys Val Th - #r Leu    #           285    - Ala Glu Gln Asp Pro Asp Glu Leu Asn Lys Al - #a Cys    #   300    - Ser Phe Ser Lys Pro Ser Asn Ser Trp Phe Pr - #o Val    #               310    - Glu Gly Pro Ala Asp Ile Cys Gln Cys Cys As - #n Lys    #       320    - Gly Asp Cys Gly Thr Pro Ser His Ser Arg Ar - #g Gln    325                 3 - #30                 3 - #35    - Pro His Val Met Ser Gln Trp Ser Arg Ser Al - #a Ser    #           345    - Arg Asn Arg Arg His Val Thr Glu Glu Ala As - #p Val    #   360    - Thr Val Gly Pro Leu Ile Phe Leu Asp Arg Ar - #g Gly    #               370    - Asp His Glu Val Glu Gln Trp Ala Leu Pro Se - #r Asp    #       380    - Thr Ser Val Val Leu Leu Gly Val Gly Leu Al - #a Val    385                 3 - #90                 3 - #95    - Val Val Ser Leu Thr Leu Thr Ala Val Ile Le - #u Val    #           405    - Leu Thr Arg Arg Cys Arg Thr Ala Ser His Pr - #o Val    #   420    - Ser Ala Ser Glu    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  424              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  unknown    -     (ii) MOLECULE TYPE: protein    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: mouse              (B) STRAIN:              (C) INDIVIDUAL ISOLATE:              (D) DEVELOPMENTAL STAGE:              (E) HAPLOTYPE:              (F) TISSUE TYPE:              (G) CELL TYPE:              (H) CELL LINE:              (I) ORGANELLE:    -     (ix) FEATURE:              (A) NAME/KEY: ZP3              (B) LOCATION:              (C) IDENTIFICATION METHOD:    #mouse ZP3 proteinR INFORMATION:    -     (xi) SEQUENCE DESCRIPTION:SEQ  ID NO:8:    - Met Ala Ser Ser Tyr Phe Leu Phe Leu Cys Le - #u Leu    #                10    - Leu Cys Gly Gly Pro Glu Leu Cys Asn Ser Gl - #n Thr    #        20    - Leu Trp Leu Leu Pro Gly Gly Thr Pro Thr Pr - #o Val    #35    - Gly Ser Ser Ser Pro Val Lys Val Glu Cys Le - #u Glu    #            45    - Ala Glu Leu Val Val Thr Val Ser Arg Asp Le - #u Phe    #    60    - Gly Thr Gly Lys Leu Val Gln Pro Gly Asp Le - #u Thr    #                70    - Leu Gly Ser Glu Gly Cys Gln Pro Arg Val Se - #r Val    #        80    - Asp Thr Asp Val Val Arg Phe Asn Ala Gln Le - #u His    #95    - Glu Cys Ser Ser Arg Val Gln Met Thr Lys As - #p Ala    #           105    - Leu Val Tyr Ser Thr Phe Leu Leu His Asp Pr - #o Arg    #   120    - Pro Val Ser Gly Leu Ser Ile Leu Arg Thr As - #n Arg    #               130    - Val Glu Val Pro Ile Glu Cys Arg Tyr Pro Ar - #g Gln    #       140    - Gly Asn Val Ser Ser His Pro Ile Gln Pro Th - #r Trp    145                 1 - #50                 1 - #55    - Val Pro Phe Arg Ala Thr Val Ser Ser Glu Gl - #u Lys    #           165    - Leu Ala Phe Ser Leu Arg Leu Met Glu Glu As - #n Trp    #   180    - Asn Thr Glu Lys Ser Ala Pro Thr Phe His Le - #u Gly    #               190    - Glu Val Ala His Leu Gln Ala Glu Val Gln Th - #r Gly    #       200    - Ser His Leu Pro Leu Gln Leu Phe Val Asp Hi - #s Cys    205                 2 - #10                 2 - #15    - Val Ala Thr Pro Ser Pro Leu Pro Asp Pro As - #n Ser    #           225    - Ser Pro Tyr His Phe Ile Val Asp Phe His Gl - #y Cys    #   240    - Leu Val Asp Gly Leu Ser Glu Ser Phe Ser Al - #a Phe    #               250    - Gln Val Pro Arg Pro Arg Pro Glu Thr Leu Gl - #n Phe    #       260    - Thr Val Asp Val Phe His Phe Ala Asn Ser Se - #r Arg    265                 2 - #70                 2 - #75    - Asn Thr Leu Tyr Ile Thr Cys His Leu Lys Va - #l Ala    #           285    - Pro Ala Asn Gln Ile Pro Asp Lys Leu Asn Ly - #s Ala    #   300    - Cys Ser Phe Asn Lys Thr Ser Gln Ser Trp Le - #u Pro    #               310    - Val Glu Gly Asp Ala Asp Ile Cys Asp Cys Cy - #s Ser    #       320    - His Gly Asn Cys Ser Asn Ser Ser Ser Ser Gl - #n Phe    325                 3 - #30                 3 - #35    - Gln Ile His Gly Pro Arg Gln Trp Ser Lys Le - #u Val    #           345    - Ser Arg Asn Arg Arg His Val Thr Asp Glu Al - #a Asp    #   360    - Val Thr Val Gly Pro Leu Ile Phe Leu Gly Ly - #s Ala    #               370    - Asn Asp Gln Thr Val Glu Gly Trp Thr Ala Se - #r Ala    #       380    - Gln Thr Ser Val Ala Leu Gly Leu Gly Leu Al - #a Thr    385                 3 - #90                 3 - #95    - Val Ala Phe Leu Thr Leu Ala Ala Ile Val Le - #u Ala    #           405    - Val Thr Arg Lys Cys His Ser Ser Ser Tyr Le - #u Val    #   420    - Ser Leu Pro Gln    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  16              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  unknown    -     (ii) MOLECULE TYPE: peptide    -     (ix) FEATURE:              (A) NAME/KEY:              (B) LOCATION:              (C) IDENTIFICATION METHOD:    #Human ZP2 epitopeR INFORMATION:                   from amin - #o acids 118-113 of human ZP2.    -     (xi) SEQUENCE DESCRIPTION:SEQ  ID NO:9:    - Ile Arg Val Met Asn Asn Ser Ala Ala Leu Ar - #g His    #                10    - Gly Ala Val Met             15    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  16              (B) TYPE:  amino aci - #ds              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  unknown    -     (ii) MOLECULE TYPE: peptide    -     (ix) FEATURE:              (A) NAME/KEY:              (B) LOCATION:              (C) IDENTIFICATION METHOD:              (D) OTHER INFORMATION:mous - #e ZP2 epitope from                   amino aci - #ds 114-129 of mouse ZP2 protein.    -     (xi) SEQUENCE DESCRIPTION:SEQ  ID NO:10:    - Ile Arg Val Gly Asp Thr Thr Thr Asp Val Ar - #g Tyr    #                10    - Lys Asp Asp Met            15    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  16              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  unknown    -     (ix) FEATURE:              (A) NAME/KEY:              (B) LOCATION:              (C) IDENTIFICATION METHOD:    #human ZP3 epitopeR INFORMATION:                   from amin - #o acids 327-342 of human ZP3 protein.    -     (ii) MOLECULE TYPE:    -     (xi) SEQUENCE DESCRIPTION:SEQ  ID NO:11:    - Cys Gly Thr Pro Ser His Ser Arg Arg Gln Pr - #o His    #                10    - Val Met Ser Gln            15    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  16              (B) TYPE:  amino aci - #d              (C) STRANDEDNESS:  sing - #le              (D) TOPOLOGY:  unknown    -     (ii) MOLECULE TYPE: peptide    -     (ix) FEATURE:              (A) NAME/KEY:              (B) LOCATION:              (C) IDENTIFICATION METHOD:    #Mouse ZP3 epitopeR INFORMATION:                   from amin - #o acids 328-343 of mouse ZP3 protein.    -     (xi) SEQUENCE DESCRIPTION:SEQ  ID NO:12:    - Cys Ser Asn Ser Ser Ser Ser Gln Phe Gln Il - #e His    #                10    - Gly Pro Arg Gln            15    __________________________________________________________________________

What is claimed is:
 1. An isolated DNA segment encoding the mouse ZP2protein having the SEQ ID No.6.
 2. An isolated DNA segment encoding thehuman ZP2 protein having the SEQ ID No.5.
 3. A recombinant DNA vectorcomprising the DNA segment according to claim
 2. 4. A culture of cellstransformed with said recombinant DNA vector according to claim
 3. 5. Amethod of producing at least a portion of a human ZP2 protein comprisingculturing cells according to claim 4 under conditions such that saidprotein is produced and isolating said protein from culture media orfrom said cells.
 6. A purified protein encoded by said DNA segment ofclaim 2, said protein having the amino acid sequence shown in FIG. 4.